Methods for treating, ameliorating, or preventing viral infections

ABSTRACT

The present disclosure relates, in certain embodiments, to the finding that certain compounds that modulate the activity(ies) of Sigma receptors can be used to disrupt virus lifecycle, infection, and/or dissemination. The compounds contemplated in the disclosure are useful in the treatment or amelioration of virus infection, such as but not limited to coronavirus infection, either alone or in combination with at least one additional therapeutic agent, which can be an antiviral agent and/or an agent that treats, ameliorates, and/or prevents one or more virus infection symptoms or co-morbidities. In certain embodiments, the Sigma receptor is a Sigma-1 receptor (also known as Sigma 1) or Sigma-2 receptor (also known as Sigma2 or TMEM97).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. No. 63/001,900 filed Mar. 30, 2020, thecontent of which is incorporated by reference herein in its entirety.

BACKGROUND

In eukaryotic cells the endoplasmic reticulum (ER) is the primary siteof synthesis, folding, and assembly of secreted and integral membraneproteins and their macromolecular complexes. Maintenance of ER proteinhomeostasis relies on the timely convergence of multiple pathways thatdetect homeostatic protein concentration thresholds and control theebb-and-flow of ER proteins. This process is driven by an intricatenetwork of molecular chaperones and transcription factors. Disruption ofER homeostasis activates stress response pathways, including theunfolded protein response (UPR).

The mammalian UPR comprises at least two phases: an initial alarm phase,which is followed by a cytoprotective, adaptive phase in which UPRfactors are upregulated to enhance the cellular capacity to processincreased concentrations of unfolded protein. Imbalanced or alteredcapacity to respond to ER stress has been implicated in various diseasesand disorders. Protracted ER stress can overwhelm the UPR, leading toautophagy as a secondary survival response. Although the relationshipbetween ER stress, unfolded protein response, and autophagy remainsunclear, growing evidence suggests that these responses are likelyintegrated signaling pathways that modulate cell survival and growth.

Autophagy describes a set of bulk cellular degradation pathways in whichlarge aggregates of misfolded proteins and damaged cellular components,including damaged organelles, are sequestered into membrane boundvesicles called autophagosomes and subsequently targeted for lysosomaldegradation. Complete autophagy comprises autophagosome fusion withlysosomes to form autolysosomes, wherein the sequestered proteins andlipids are subsequently degraded by autophagic degradation or flux.Autophagy occurs under basal conditions in many tissues and is involvedin cellular differentiation and development. It is also (hyper)activatedin conditions of nutrient starvation and cellular stress, to maintainenergy levels and to sequester and remove damaged and cytotoxic cellularcomponents. Thus, autophagy plays important roles in cellularhomeostasis and disease prevention, and defective autophagy has beenimplicated in neurodegenerative disease and cancer.

Sigma receptors are distinct from classical opioid receptors. Sigma1 ishighly conserved among mammals (greater than 80% amino acid identity),but shares no significant homology with any traditional receptor familyor other mammalian protein. Cloned Sigma1 is a 26 kilodalton integralmembrane protein found primarily in the ER, and can translocate to theplasma membrane, other organelles, and endoplasmic membranemicrodomains. Recent work has described Sigma ligand-induced cell deathby lysosomal destabilization and oxidative stress.

There is thus a need in the art to identify compounds useful in thetreatment of viral infections, such as but not limited to coronavirusinfections, such as but not limited to SARS-CoV and/or SARS-CoV-2. Thepresent disclosure addresses this unmet need.

BRIEF SUMMARY

The present disclosure provides a method of disrupting virus lifecycle,infection, and/or dissemination in a virus-infected subject, and/orpreventing and/or minimizing virus infection and/or dissemination in asubject.

The present disclosure provides a method of inhibiting, minimizing,and/or preventing formation of virus particles in a virus-infectedsubject.

The present disclosure provides a method of altering production,post-translational modification, assembly, maturation, and/or functionalcell surface expression of at least one virus protein involved in viralentry and/or viral infection in a subject's cell.

The present disclosure provides a method of initiating and/orstimulating selective autophagosomal, lysosomal, and/or proteasomaldegradation of at least one virus protein involved in viral entry and/orviral infection in a subject's cell.

The present disclosure provides a method of decreasing and/or inhibitingincrease of amount, concentration, and/or production of at least onevirus protein involved in viral entry and/or viral infection in asubject's cell.

The present disclosure provides a method of altering and/or disturbingsubcellular localization and/or virus promoting activity of at least onevirus protein involved in viral entry and/or viral infection in asubject's cell.

The present disclosure provides a method of minimizing and/or preventingincorporation of a surface viral protein into a virion in avirus-infected eukaryotic cell.

In certain embodiments, the virus comprises at least one surface viralprotein that is involved in viral entry and/or viral infection.

In certain embodiments, the method comprises administering to thesubject a therapeutically effective amount of a Sigma1inhibitor/antagonist that causes and/or triggers ER stress and/orautophagy in the subject.

In certain embodiments, the method comprises administering to thesubject a therapeutically effective amount of a compound of Formula (I),as described elsewhere herein, or a salt, solvate, or N-oxide thereof;and any combinations thereof.

In certain embodiments, the method comprises administering to thesubject a therapeutically effective amount of a compound of Formula(II), as described elsewhere herein, or a salt, solvate, or N-oxidethereof; and any combinations thereof.

In certain embodiments, the method comprises administering to thesubject a therapeutically effective amount of a compound of Formula(III), as described elsewhere herein, or a salt, solvate, or N-oxidethereof; and any combinations thereof.

In certain embodiments, the virus comprises a flavivirus.

In certain embodiments, the flavivirus comprises at least one of Apoivirus, Aroa virus, Bamaga virus, Bagaza virus, Banzi virus, Boubouivirus, Bukalasa bat virus, Cacipacore virus, Carey Island virus, CowboneRidge virus, Dakar bat virus, Dengue virus, Edge Hill virus, Entebbe batvirus, Gadgets Gully virus, Ilheus virus, Israel turkeymeningoencephalomyelitis virus, Japanese encephalitis virus, Jugravirus, Jutiapa virus, Kadam virus, Kedougou virus, Kokobera virus,Koutango virus, Kyasanur Forest disease virus, Langat virus, Louping illvirus, Meaban virus, Modoc virus, Montana myotis leukoencephalitisvirus, Murray Valley encephalitis virus, Ntaya virus, Omsk hemorrhagicfever virus, Phnom Penh bat virus, Powassan virus, Rio Bravo virus,Royal Farm virus, Saboya virus, Saint Louis encephalitis virus, SalVieja virus, San Perlita virus, Saumarez Reef virus, Sepik virus,Tembusu virus, Tick-borne encephalitis virus, Tyuleniy virus, Uganda Svirus, Usutu virus, Wesselsbron virus, West Nile virus, Yaounde virus,Yellow fever virus, Yokose virus, and Zika virus.

In certain embodiments, the virus comprises a Coronavirus.

In certain embodiments, the Coronavirus comprises at least one of anAlphacoronavirus, a Betacoronavirus, a Gammacoronavirus, and aDeltacoronavirus.

In certain embodiments, the Coronavirus comprises at least one ofMERS-CoV, SARS-CoV, and SARS-CoV-2.

In certain embodiments, the virus is a Coronavirus and wherein at leastone coronavirus protein comprises NSP6 and/or a S glycoprotein.

In certain embodiments, the compound is selected from the groupconsisting of: 1-(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine(Compound A); 1-(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine(Compound B); 1-(n-propyl)-3-(4-iodophenyl)guanidine (Compound C);1-(n-propyl)-3-(4-methoxyphenyl)guanidine (Compound D);1,3-bis(3-(4-fluorophenoxy)propyl)guanidine (Compound E);1-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine(Compound F); 1-(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine(Compound G);1-(3-(4-fluorophenoxy)propyl)-3-(4-methyl-2-oxo-2H-chromen-7-yl)guanidine)(Compound H); a salt, solvate or N-oxide thereof, and any combinationsthereof.

In certain embodiments, the compound is administered as a pharmaceuticalcomposition further comprising a pharmaceutically acceptable carrier.

In certain embodiments, the subject is further administered at least oneadditional antiviral agent and/or at least one agent that treats one ormore virus infection symptoms or co-morbidities.

In certain embodiments, the compound is administered by a routecomprising oral, nasal, rectal, intravaginal, parenteral, buccal,sublingual or topical.

In certain embodiments, the subject is a mammal.

In certain embodiments, the mammal is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of specific embodiments of thedisclosure will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the disclosure,exemplary embodiments are shown in the drawings. It should beunderstood, however, that the disclosure is not limited to the precisearrangements and instrumentalities of the embodiments shown in thedrawings.

FIGS. 1A-1D illustrate the finding that Sigma1 modulators suppressTMPRSS2 mRNA transcript levels in part by allosteric inhibition ofandrogen receptor (AR). FIG. 1A: TMPRSS2 transcript levels decrease inSigma1 siRNA (siR) mediated knockdown LNCaP cells. FIG. 1B: TMPRSS2transcript decreased in LNCaP cells treated with Sigma1 modulator IPAG(also known as 1-(4-iodophenyl)-3-(2-adamantyl)guanidine; 10 μM). FIGS.1C-1D: AR luciferase reporter assay shows inhibition of dihydrotesterone(DHT) induced AR transcriptional activity by IPAG (FIG. 1C), CT189 (alsoknown as 1-(4-chlorophenyl)-3-(3-(4-fluorophenoxy)-prop-1-yl)guanidine;FIG. 1D).

FIGS. 2A-2D illustrate the finding that Sigma1 is expressed in lungs andSIGMAR1 mRNA levels are enriched in alveolar type II (AT2) epithelialcells and correlate with ACE2 and TMPRSS2 transcript levels. FIG. 2A:Sigma1 protein expression in lung tissue. IHC performed with anti-Sigma1antibody (described in Schrock et al, 2013, Mol Pharmacol 84:751-762;and Thomas et al, 2017, Cancer Research, DOI: 10.1158/0008-5472.CAN-16-1055). Inset of indicated area shown below. FIG. 2B: SIGMAR1transcript levels in subpopulations of lung cells: Alveolar type I (AT1)and type II (AT2) epithelial cells, Ciliated cells, club cells,epithelial cells, fibroblasts, immune (monocytes, T-cells) and lymphaticendothelial (Lymph.Endo.) cells. Analysis was performed using datasetpublished in Lukassen et al, 2020, The EMBO J. e105114). CPM summedacross each cell type per patient. FIG. 2C: Pearson correlation ofSIGMAR1 and TMPRSS2 and ACE2 mRNA in lung AT2 cells. FIG. 2D: Pearsoncorrelation of ACE2 and TMPRSS2 in lung AT2 cells.

FIG. 3 illustrates the finding that only certain Sigma1 inhibitors blockSARS-CoV-2 infection. Infection of Huh7.5 cells by replication competentSARS-CoV-2 in the presence of 10 μM Sigma1 Inhibitor/antagonist—IPAG((also known as 1-(4-iodophenyl)-3-(2-adamantyl)guanidine), CT189 (alsoknown as 1-(4-chlorophenyl)-3-(3-(4-fluorophenoxy)-prop-1-yl)guanidine),S1RA (also known as4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl]morpholine),haloperidol (also known as4-[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]-1-(4-fluorophenyl)butan-1-one;or Hal)—or 10 μM Sigma1 Activator/agonist: SA4503 (also known as1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl) piperazine), PRE084 (alsoknown as 2-morpholin-4-ylethyl 1-phenylcyclohexane-1-carboxylate).Results can be compared to vehicle (DMSO), Remdesivir (Rem, 10 PM), andhydroxychloroquine (HCQ, 10 μM). Compounds and SARS-CoV-2 were added tocell culture; 24 hours post inoculation relative infection calculated byqRT-PCR.

FIG. 4 illustrates results obtained when Huh7.5 cells were treated withcompounds at 10 μM for 2 h, then infected with SARS-CoV-2 for 24 h.Lysates were probed with α-spike and α-β actin antibodies. Western blotis representative of two experimental replicates.

FIG. 5 illustrates the finding that SARS-CoV-2 nucleocapsid protein isdetected in Syrian golden hamster lungs at 3 days p.i. but is absent inuninfected hamsters.

FIG. 6 illustrates the finding that compounds of the disclosure inhibitDengue virus infection. U20S cells were treated with DMSO and 10 μM ofCT110 (also known as CT189). 2 hours after starting treatment, U20Scells were infected with a multiplicity of infection (MOI) of 1 forDengue virus and incubated at 37° C. for ˜16-18 hours. Cells wereharvested with Trizol and RNA was extracted for qPCR. Y-axis indicatesfold change in infection.

FIG. 7 illustrates the finding that compounds of the disclosure inhibitZika virus infection. U20S cells were treated with DMSO and 10 μM ofCT110 (also known as CT189). 2 hours after starting treatment, U20Scells were infected with a multiplicity of infection (MOI) of 1 for Zikavirus and incubated at 37° C. for ˜16-18 hours. Cells were harvestedwith Trizol and RNA was extracted for qPCR. Y-axis indicates fold changein infection.

FIG. 8 illustrates the finding that compounds of the disclosure inhibitPowassan virus infection. U20S cells were treated with DMSO and 10 μM ofCT110 (also known as CT189). 2 hours after starting treatment, U20Scells were infected with a multiplicity of infection (MOI) of 1 forPowassan virus and incubated at 37° C. for ˜16-18 hours. Cells wereharvested with Trizol and RNA was extracted for qPCR. Y-axis indicatesfold change in infection.

DETAILED DESCRIPTION

The present disclosure relates to the finding that certain compoundsthat bind to and inhibit activity of the Sigma receptor modulators canbe used to disrupt viral lifecycle, infection, and/or dissemination. Thecompounds contemplated in the disclosure are useful in the treatment oramelioration of viral infection, either alone or in combination with atleast one additional therapeutic agent, which can be an antiviral agentand/or an agent that treats, ameliorates, and/or ameliorates one or moreviral infection symptoms or co-morbidities. In other embodiments, theSigma receptor is a Sigma1 receptor (also known as Sigma1).

In certain embodiments, the virus comprises a flavivirus. In otherembodiments, the flavivirus comprises at least one of Zika virus, Denguevirus, and Powassan virus. In yet other embodiments, the flaviviruscomprises at least one of Apoi virus, Aroa virus, Bamaga virus, Bagazavirus, Banzi virus, Bouboui virus, Bukalasa bat virus, Cacipacore virus,Carey Island virus, Cowbone Ridge virus, Dakar bat virus, Dengue virus,Edge Hill virus, Entebbe bat virus, Gadgets Gully virus, Ilheus virus,Israel turkey meningoencephalomyelitis virus, Japanese encephalitisvirus, Jugra virus, Jutiapa virus, Kadam virus, Kedougou virus, Kokoberavirus, Koutango virus, Kyasanur Forest disease virus, Langat virus,Louping ill virus, Meaban virus, Modoc virus, Montana myotisleukoencephalitis virus, Murray Valley encephalitis virus, Ntaya virus,Omsk hemorrhagic fever virus, Phnom Penh bat virus, Powassan virus, RioBravo virus, Royal Farm virus, Saboya virus, Saint Louis encephalitisvirus, Sal Vieja virus, San Perlita virus, Saumarez Reef virus, Sepikvirus, Tembusu virus, Tick-borne encephalitis virus, Tyuleniy virus,Uganda S virus, Usutu virus, Wesselsbron virus, West Nile virus, Yaoundevirus, Yellow fever virus, Yokose virus, and Zika virus.

In certain embodiments, the virus comprises a Coronavirus. In otherembodiments, the Coronavirus comprises at least one of anAlphacoronavirus, a Betacoronavirus, a Gammacoronavirus, and aDeltacoronavirus. In yet other embodiments, the Coronavirus comprises atleast one of MERS-CoV, SARS-CoV, and SARS-CoV-2, and any variantsthereof.

Sigma1 (gene name SIGMAR1; also known as sigma1-receptor) is a uniquepharmacologically-responsive intracellular integral membrane scaffoldingprotein. Sigma1 is enriched in the secretory pathway, particularly theendoplasmic reticulum (ER) of most cells. Sigma1 itself has no knownintrinsic signaling or enzymatic activity, rather it allostericallymodulates the intracellular signaling and activities of its associatedproteins. Sigma1 physically associates with multiple integral membraneproteins and functions via protein-protein interactions to regulatetheir stability, transport, and degradation in a pharmacologicallycontrollable manner. The multifunctionality of Sigma1 enables it toregulate lipid and protein homeostasis at multiple levels and it plays acritical role in protein synthesis, processing, trafficking, assembly,and quality control in the secretory pathway of cells. Thus, targetingSigma1 enables selective modulation of multiple cellular processes viaone drug target. Certain Sigma1 modulators can block glycosylation,maturation, and transport of integral membrane glycoproteins in the ER,block their cell surface expression, and trigger lysosomal degradation.In certain embodiments, these features of Sigma1 modulators can beexploited to suppress viral proteins in infected cells and/or suppressviral replication and/or suppress production of viral particles. Incertain embodiments, certain Sigma1 modulators can be used to minimizeand/or reduce viral load and/or block viral dissemination.

The on-target actions of Sigma1 modulators do not induce adverseeffects. Additionally, SIGMAR1 knockout (KO) mice are viable and do notdisplay a phenotype overtly different from wild type mice, indicatingthat Sigma1 inhibition has minimal impact on normal tissues.

In certain embodiments, Sigma1 inhibitors/antagonists that causa and/ortrigger ER stress and/or autophagy are useful within the methods of thedisclosure. In certain embodiments, certain Sigma1inhibitors/antagonists can suppress viral infection, replication, andsubsequent cytopathic effects (CPE). In certain embodiments, certainSigma1 inhibitors/antagonists can suppress SARS-CoV-2 infection,replication, and subsequent cytopathic effects (CPE). In certainembodiments, certain Sigma1 inhibitors/antagonists can suppress Sprotein maturation and trigger its degradation via ER stress associatedproteasomal or lysosomal mechanisms, thus preventing S proteinincorporation into SARS-CoV-2 virions and/or suppressing TMPRSS2 proteinlevels and cell surface localization on susceptible host cells. Incertain embodiments, certain Sigma1 inhibitors/antagonists can suppressmaturation of at least one surface viral protein and trigger itsdegradation via ER stress associated proteasomal or lysosomalmechanisms, thus preventing incorporation of the at least one surfaceprotein into the virion. In certain embodiments, certain Sigma1inhibitors/antagonists induce degradation of a surface viral proteinwhich is key for viral infection, and this results in reduced orweakened viral infection and/or reduced viral loads. In certainembodiments, certain Sigma1 inhibitor/antagonist-induced degradation ofSARS-CoV-2 S protein and suppression of TMPRSS2 expression can restrictSARS-CoV-2 infection, reduced viral loads, and/or reduced pathology inthe lungs of animals treated with certain Sigma1 inhibitors/antagonists.

In one aspect, any embodiment described herein is not limited toSARS-CoV-2, and is also applicable to other viruses that use a similarroute of entry and mechanism of replication as SARS-Cov-2, such asviruses that utilize at least one surface viral protein, such as Spike,as a means to penetrate a host cell.

As described herein, Sigma1 and Sigma2 targeting therapies can be usedto treat infection by severe acute respiratory syndrome coronavirus(SARS-CoV and/or SARS-CoV-2) and related infectious agents. In certainembodiments, the compounds contemplated in the disclosure can be used totreat or ameliorate infection caused by SARS-CoV and/or SARS-CoV-2 andrelated infection agents without promoting undesirable and/ortreatment-limiting central nervous system (CNS)-related side effects.

In certain embodiments, the compounds contemplated in the disclosuretrigger pathways that regulate protein and lipid homeostasis in thesecretory pathway (endoplasmic reticulum, Golgi, plasma membrane, andassociated vesicles including lysosomes, autolysosomes, andendolysosomes). The compounds contemplated in the disclosure, but notall compounds with affinity for Sigma, can be used to selectivelyregulate protein trafficking, production, and degradation. Thesecompounds contemplated in the disclosure do so in part by triggering theunfolded protein response (UPR) and selective autophagy. Such biologicalmodulations are useful in treating or ameliorating infection caused bySARS-CoV, SARS-Cov-2, and related infectious agents.

In certain embodiments, Sigma1 physically interacts with the SARS-CoV-2protein Non-Structural protein 6 (NSP6), which is a membrane-spanningintegral component of the SARS-CoV-2 viral replication complex that isimplicated in double membrane vesicle (DMV) formation. In certainembodiments, SARS-CoV-2 protein NSP6 plays a role in regulation ofautophagy of the infected cell. However, at the time of the presentdisclosure there was no knowledge whether compounds with affinity forSigma1 and Sigma2 would have any activity in the SARS-CoV-2 diseasemodel: it was unclear which Sigma targeted compounds would beefficacious in treating SARS-CoV-2 and which would bind to Sigma1 and/orSigma2 and demonstrate actual efficacy in the disease model. In certainembodiments, a compound useful within the present methods triggers UPR,autophagy, and lysosomal mechanisms via Sigma receptors, and yet haslimited and/or manageable side effects, which do not limit their utilityas therapeutic agents. In certain embodiments, the compoundscontemplated within the disclosure induce the UPR, autophagy, andlysosomal mechanisms as described elsewhere herein, but with improvedsafety and efficacy.

In certain embodiments, the compounds contemplated within the disclosurepromote autophagic degradation of NSP6 in a coronavirus-infected cell.In certain embodiments, the compounds contemplated within the disclosuredecrease or inhibit increase of NSP6 concentration in acoronavirus-infected cell and/or alter the subcellular localizationand/or virus promoting activity of NSP6. In certain embodiments, thecompounds contemplated within the disclosure inhibit, minimize, orprevent formation of new coronavirus particles in a coronavirus-infectedcell. In certain embodiments, the compounds contemplated within theinvention alter production, post-translational modification, assembly,and functional cell surface expression of proteins involved in host cellviral entry and/or host cell infection such as but not limited to NSP6and/or a coronavirus spike (S) glycoprotein. In certain embodiments, thecompounds contemplated within the invention trigger the selectiveautophagosomal, lysosomal, and proteasomal degradation of coronavirusproteins including NSP6 and/or a spike (S) glycoprotein in the hostcell. In certain embodiments, the compounds contemplated within theinvention suppress protein production (such as but not limited to NSP6and/or a S glycoprotein) in the host cell.

In certain embodiments, activities of compounds contemplated within theinvention can be assessed using one or more of the following assays: (1)viral infectivity measured by plaque assay; (2) viral replication andproduction assay by virus isolation and protein and nucleotide analysis(methods similar to current RT-PCR based detection methods); (3) cellbased assay measuring NSP6 levels and/or localization; (4) cell basedand biochemical assays measuring induction (formation of autophagosomes)and completion of autophagy (autophagic flux/degradation of cargo)mediated by NSP6; (5) cell-to-cell fusion assay with recombinant Sprotein expressing cells; (6) syncytium assay with recombinant S proteinexpressing cells; (7) NSP6 and S glycoprotein levels by immunoblotand/or fluorescent and/or luciferase tagged recombinant versions ofthese proteins; (8) protein localization by subcellular fractionationand immunoblot, confocal imaging of fluorescence tagged protein oruntagged proteins by immunocytochemistry/fluorescence microscopy; (9)pseudovirus infection assay with SARS-CoV-2 S pseudovirions to measurevirus entry; (10) correlation of results from (1)-(9) to “connect thedots” to determine which Sigma modulator impacted activities correlatewith decreased infectivity.

Coronaviruses are entirely dependent on the host's cellular machinery toreplicate. In certain embodiments, Sigma1 targeted agents can be used toprevent SARS-CoV-2 from co-opting the host cell's machinery to replicateand produce new infectious viral particles. In certain embodiments,Sigma1 modulators can disrupt two proteins crucial to infection andreplication: Spike (S) protein (targeting the virus) and TMPRSS2(modifying the host cell). S protein is a SARS-CoV-2 integral membraneglycoprotein that enables SARS-CoV-2 to recognize and infect host cells.TMPRSS2 is a cellular membrane glycoprotein that acts as a cofactor toangiotensin converting enzyme 2 (ACE2), the cellular receptor recognizedby S protein. TMPRSS2 enhances infection by cleaving and thus activatingthe S protein to trigger membrane fusion that enables viral entry intohost cells. Elevated TMPRSS2 on host cells dramatically increasessusceptibility to SARS-CoV-2 infection.

The present disclosure includes any compound contemplated within thedisclosure, as well as compositions comprising the same, wherein thecompositions optionally further comprise at least one additionaltherapeutic agent and/or at least one pharmaceutically acceptablecarrier.

In certain embodiments, the compounds contemplated within the disclosurehave improved drug-like properties over compounds known in the art tobind to and modulate the Sigma receptor. In other embodiments, thecompounds contemplated within the disclosure do not cross theblood-brain barrier. In yet other embodiments, the compoundscontemplated within the disclosure cross the blood-brain barrier.

The compounds contemplated within the disclosure can be characterized bypharmacological, cellular, biochemical, in vivo, pharmacokinetics, orpharmacodynamics properties. Selected examples of characterizationstudies include, but are not limited to, Sigma1-ligand bindingproperties, signaling pathway analysis and/or characterization,proteomic analysis of Sigma1 protein associations in response to Sigmaligand treatment, tumor, brain response, and toxicity.

The entire disclosures of WO2014/015157 and US 2015/0166472 areincorporated herein in their entireties by reference.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of 20% or ±10%, more preferably +5%, even more preferably10%, and still more preferably +0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

The term “abnormal,” when used in the context of organisms, tissues,cells or components thereof, refers to those organisms, tissues, cellsor components thereof that differ in at least one observable ordetectable characteristic (e.g., age, treatment, time of day, etc.) fromthose organisms, tissues, cells or components thereof that display the“normal” (expected) respective characteristic. Characteristics that arenormal or expected for one cell or tissue type might be abnormal for adifferent cell or tissue type.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate.

In contrast, a “disorder” in an animal is a state of health in which theanimal is able to maintain homeostasis, but in which the animal's stateof health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

A disease or disorder is “alleviated” or “ameliorated” if the severityof a symptom of the disease or disorder, the frequency with which such asymptom is experienced by a patient, or both, is reduced.

As used herein, the term “Sigma” refers to the Sigma1 receptor (Sigma1),Sigma2 receptor (Sigma2), any splice variant thereof or any isoformthereof.

As used herein, a “Sigma receptor modulator” is a compound that binds tothe Sigma receptor and modifies the activity or biological function ofthe receptor as compared to the activity or biological function of thereceptor in the absence of the modulator. In certain embodiments, themodulator can activate the receptor and thus cause a biological responsethat is enhanced over the baseline activity of the unbound receptor. Incertain embodiments, the modulator cannot activate the receptorthoroughly and thus causes a biological response that is smaller inmagnitude compared to those of full modulators. In certain embodiments,the modulator can bind to the receptor but does not activate it,resulting in receptor blockage and inhibiting the binding of othermodulators. Such an modulator does not diminish the baselineintracellular response in the absence of an modulator. In certainembodiments, the modulator can function as a putative antagonist,agonist, or as an inverse agonist, which reduces the activity of thereceptor by inhibiting its constitutive activity.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In a non-limiting embodiment, the patient, subject or individual is ahuman.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology, for the purpose of diminishing oreliminating those signs.

As used herein, the term “treatment” or “treating” is defined as theapplication or administration of a therapeutic agent, i.e., a compoundof the disclosure (alone or in combination with another pharmaceuticalagent), to a patient, or application or administration of a therapeuticagent to an isolated tissue or cell line from a patient (e.g., fordiagnosis or ex vivo applications), who has a condition contemplatedherein and/or a symptom of a condition contemplated herein, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect a condition contemplated herein and/or the symptoms ofa condition contemplated herein. Such treatments may be specificallytailored or modified, based on knowledge obtained from the field ofpharmacogenomics.

As used herein, the term “composition” or “pharmaceutical composition”refers to a mixture of at least one compound useful within thedisclosure with a pharmaceutically acceptable carrier. Thepharmaceutical composition facilitates administration of the compound toa patient or subject. Multiple techniques of administering a compoundexist in the art including, but not limited to, intravenous, oral,aerosol, parenteral, ophthalmic, pulmonary and topical administration.

The phrase “therapeutically effective amount,” as used herein, refers toan amount that is sufficient or effective to prevent or treat (delay orprevent the onset of, prevent the progression of, inhibit, decrease orreverse) a disease or condition associated with the Sigma receptor,including alleviating symptoms of such diseases.

As used herein, the terms “effective amount,” “pharmaceuticallyeffective amount” and “therapeutically effective amount” refer to anontoxic but sufficient amount of an agent to provide the desiredbiological result. That result may be reduction and/or alleviation ofthe signs, symptoms, or causes of a disease, or any other desiredalteration of a biological system.

An appropriate therapeutic amount in any individual case may bedetermined by one of ordinary skill in the art using routineexperimentation.

An “effective amount” of a delivery vehicle is that amount sufficient toeffectively bind or deliver a compound.

As used herein, the term “potency” refers to the dose needed to producehalf the maximal response (ED₅₀).

As used herein, the term “efficacy” refers to the maximal effect(E_(max)) achieved within an assay.

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound, and is relativelynon-toxic, i.e., the material may be administered to an individualwithout causing undesirable biological effects or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

As used herein, the language “pharmaceutically acceptable salt” refersto a salt of the administered compounds prepared from pharmaceuticallyacceptable non-toxic acids, including inorganic acids, organic acids,solvates, hydrates, or clathrates thereof. Examples of such inorganicacids are hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric,phosphoric, acetic, hexafluorophosphoric, citric, gluconic, benzoic,propionic, butyric, sulfosalicylic, maleic, lauric, malic, fumaric,succinic, tartaric, amsonic, pamoic, p-tolunenesulfonic, and mesylic.Appropriate organic acids may be selected, for example, from aliphatic,aromatic, carboxylic and sulfonic classes of organic acids, examples ofwhich are formic, acetic, propionic, succinic, camphorsulfonic, citric,fumaric, gluconic, isethionic, lactic, malic, mucic, tartaric,para-toluenesulfonic, glycolic, glucuronic, maleic, furoic, glutamic,benzoic, anthranilic, salicylic, phenylacetic, mandelic, embonic(pamoic), methanesulfonic, ethanesulfonic, pantothenic, benzenesulfonic(besylate), stearic, sulfanilic, alginic, galacturonic, and the like.Furthermore, pharmaceutically acceptable salts include, by way ofnon-limiting example, alkaline earth metal salts (e.g., calcium ormagnesium), alkali metal salts (e.g., sodium-dependent or potassium),and ammonium salts.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, stabilizer, dispersing agent, suspending agent,diluent, excipient, thickening agent, solvent or encapsulating material,involved in carrying or transporting a compound useful within thedisclosure within or to the patient such that it may perform itsintended function. Typically, such constructs are carried or transportedfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation, including thecompound useful within the disclosure, and not injurious to the patient.Some examples of materials that may serve as pharmaceutically acceptablecarriers include: sugars, such as lactose, glucose and sucrose;starches, such as corn starch and potato starch; cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients,such as cocoa butter and suppository waxes; oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; glycols, such as propylene glycol; polyols, such asglycerin, sorbitol, mannitol and polyethylene glycol; esters, such asethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; surface active agents;alginic acid; pyrogen-free water; isotonic saline; Ringer's solution;ethyl alcohol; phosphate buffer solutions; and other non-toxiccompatible substances employed in pharmaceutical formulations. As usedherein, “pharmaceutically acceptable carrier” also includes any and allcoatings, antibacterial and antifungal agents, and absorption delayingagents, and the like that are compatible with the activity of thecompound useful within the disclosure, and are physiologicallyacceptable to the patient. Supplementary active compounds may also beincorporated into the compositions. The “pharmaceutically acceptablecarrier” may further include a pharmaceutically acceptable salt of thecompound useful within the disclosure. Other additional ingredients thatmay be included in the pharmaceutical compositions used in the practiceof the disclosure are known in the art and described, for example inRemington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co.,1985, Easton, Pa.), which is incorporated herein by reference.

As used herein, the term “alkyl,” by itself or as part of anothersubstituent means, unless otherwise stated, a straight or branched chainhydrocarbon having the number of carbon atoms designated (i.e. C₁₋₆means one to six carbon atoms) and including straight, branched chain,or cyclic substituent groups. Examples include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, andcyclopropylmethyl. Most preferred is (C₁-C₆)alkyl, particularly ethyl,methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and cyclopropylmethyl.

As used herein, the term “substituted alkyl” means alkyl as definedabove, substituted by one, two or three substituents selected from thegroup consisting of halogen, —OH, alkoxy, —NH₂, —N(CH₃)₂, —C(═O)OH,trifluoromethyl, —C≡N, —C(═O)O(C₁-C₄)alkyl, —C(═O)NH₂, —SO₂NH₂,—C(═NH)NH₂, and —NO₂, preferably containing one or two substituentsselected from halogen, —OH, alkoxy, —NH₂, trifluoromethyl, —N(CH₃)₂, and—C(═O)OH, more preferably selected from halogen, alkoxy and —OH.Examples of substituted alkyls include, but are not limited to,2,2-difluoropropyl, 2-carboxycyclopentyl and 3-chloropropyl.

As used herein, the term “heteroalkyl” by itself or in combination withanother term means, unless otherwise stated, a stable straight orbranched chain alkyl group consisting of the stated number of carbonatoms and one or two heteroatoms selected from the group consisting ofO, N, and S, and wherein the nitrogen and sulfur atoms may be optionallyoxidized and the nitrogen heteroatom may be optionally quaternized. Theheteroatom(s) may be placed at any position of the heteroalkyl group,including between the rest of the heteroalkyl group and the fragment towhich it is attached, as well as attached to the most distal carbon atomin the heteroalkyl group. Examples include: —OCH₂CH₂CH₃, —CH₂CH₂CH₂OH,—CH₂CH₂NHCH₃, —CH₂SCH₂CH₃, and —CH₂CH₂S(═O)CH₃. Up to two heteroatomsmay be consecutive, such as, for example, —CH₂NHOCH₃, or —CH₂CH₂SSCH₃

As used herein, the term “alkoxy” employed alone or in combination withother terms means, unless otherwise stated, an alkyl group having thedesignated number of carbon atoms, as defined above, connected to therest of the molecule via an oxygen atom, such as, for example, methoxy,ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs andisomers. Preferred are (C₁-C₃) alkoxy, particularly ethoxy and methoxy.

As used herein, the term “halo” or “halogen” alone or as part of anothersubstituent means, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom, preferably, fluorine, chlorine, or bromine,more preferably, fluorine or chlorine.

As used herein, the term “cycloalkyl” refers to a mono cyclic orpolycyclic non-aromatic radical, wherein each of the atoms forming thering (i.e. skeletal atoms) is a carbon atom. In certain embodiments, thecycloalkyl group is saturated or partially unsaturated. In otherembodiments, the cycloalkyl group is fused with an aromatic ring.Cycloalkyl groups include groups having from 3 to 10 ring atoms.Illustrative examples of cycloalkyl groups include, but are not limitedto, the following moieties.

Monocyclic cycloalkyls include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.Dicyclic cycloalkyls include, but are not limited to,tetrahydronaphthyl, indanyl, and tetrahydropentalene. Polycycliccycloalkyls include adamantine and norbornane. The term cycloalkylincludes “unsaturated nonaromatic carbocyclyl” or “nonaromaticunsaturated carbocyclyl” groups, both of which refer to a nonaromaticcarbocycle as defined herein, which contains at least one carbon carbondouble bond or one carbon carbon triple bond.

As used herein, the term “heterocycloalkyl” or “heterocyclyl” refers toa heteroalicyclic group containing one to four ring heteroatoms eachselected from O, Sand N. In certain embodiments, each heterocycloalkylgroup has from 4 to 10 atoms in its ring system, with the proviso thatthe ring of said group does not contain two adjacent O or S atoms. Inother embodiments, the heterocycloalkyl group is fused with an aromaticring. In certain embodiments, the nitrogen and sulfur heteroatoms may beoptionally oxidized, and the nitrogen atom may be optionallyquaternized. The heterocyclic system may be attached, unless otherwisestated, at any heteroatom or carbon atom that affords a stablestructure. A heterocycle may be aromatic or non-aromatic in nature. Incertain embodiments, the heterocycle is a heteroaryl.

An example of a 3-membered heterocycloalkyl group includes, and is notlimited to, aziridine. Examples of 4-membered heterocycloalkyl groupsinclude, and are not limited to, azetidine and a beta lactam. Examplesof 5-membered heterocycloalkyl groups include, and are not limited to,pyrrolidine, oxazolidine and thiazolidinedione. Examples of 6-memberedheterocycloalkyl groups include, and are not limited to, piperidine,morpholine and piperazine. Other non-limiting examples ofheterocycloalkyl groups are.

Examples of non-aromatic heterocycles include monocyclic groups such asaziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine,pyrroline, pyrazolidine, imidazoline, dioxolane, sulfolane,2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane,piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine,morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran,1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane,4,7-dihydro-1,3-dioxepin, and hexamethyleneoxide.

As used herein, the term “aromatic” refers to a carbocycle orheterocycle with one or more polyunsaturated rings and having aromaticcharacter, i.e. having (4n+2) delocalized R (pi) electrons, where n isan integer.

As used herein, the term “aryl,” employed alone or in combination withother terms, means, unless otherwise stated, a carbocyclic aromaticsystem containing one or more rings (typically one, two or three rings),wherein such rings may be attached together in a pendent manner, such asa biphenyl, or may be fused, such as naphthalene. Examples of arylgroups include phenyl, anthracyl, and naphthyl. Preferred examples arephenyl and naphthyl, most preferred is phenyl.

As used herein, the term “aryl-(C₁-C₃)alkyl” means a functional groupwherein a one-to three-carbon alkylene chain is attached to an arylgroup, e.g., —CH₂CH₂-phenyl. Preferred is aryl-CH₂— and aryl-CH(CH₃)—.The term “substituted aryl-(C₁-C₃)alkyl” means an aryl-(C₁-C₃)alkylfunctional group in which the aryl group is substituted. Preferred issubstituted aryl(CH₂)—. Similarly, the term “heteroaryl-(C₁-C₃)alkyl”means a functional group wherein a one to three carbon alkylene chain isattached to a heteroaryl group, e.g., —CH₂CH₂-pyridyl. Preferred isheteroaryl-(CH₂)—. The term “substituted heteroaryl-(C₁-C₃)alkyl” meansa heteroaryl-(C₁-C₃)alkyl functional group in which the heteroaryl groupis substituted. Preferred is substituted heteroaryl-(CH₂)—.

As used herein, the term “heteroaryl” or “heteroaromatic” refers to aheterocycle having aromatic character. A polycyclic heteroaryl mayinclude one or more rings that are partially saturated. Examples includethe following moieties:

Examples of heteroaryl groups also include pyridyl, pyrazinyl,pyrimidinyl (particularly 2- and 4-pyrimidinyl), pyridazinyl, thienyl,furyl, pyrrolyl (particularly 2-pyrrolyl), imidazolyl, thiazolyl,oxazolyl, pyrazolyl (particularly 3- and 5-pyrazolyl), isothiazolyl,1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl,1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and1,3,4-oxadiazolyl.

Examples of polycyclic heterocycles and heteroaryls include indolyl(particularly 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl,tetrahydroquinolyl, isoquinolyl (particularly 1- and 5-isoquinolyl),1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (particularly 2-and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl,1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl,benzofuryl (particularly 3-, 4-, 5-, 6- and 7-benzofuryl),2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (particularly3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl(particularly 2-benzothiazolyl and 5-benzothiazolyl), purinyl,benzimidazolyl (particularly 2-benzimidazolyl), benzotriazolyl,thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, andquinolizidinyl.

As used herein, the term “substituted” means that an atom or group ofatoms has replaced hydrogen as the substituent attached to anothergroup. The term “substituted” further refers to any level ofsubstitution, namely mono-, di-, tri-, tetra-, or penta-substitution,where such substitution is permitted. The substituents are independentlyselected, and substitution may be at any chemically accessible position.In certain embodiments, the substituents vary in number between one andfour. In other embodiments, the substituents vary in number between oneand three. In yet other embodiments, the substituents vary in numberbetween one and two.

As used herein, the term “optionally substituted” means that thereferenced group may be substituted or unsubstituted. In certainembodiments, the referenced group is optionally substituted with zerosubstituents, i.e., the referenced group is unsubstituted. In otherembodiments, the referenced group is optionally substituted with one ormore additional group(s) individually and independently selected fromgroups described herein.

In certain embodiments, the substituents are independently selected fromthe group consisting of oxo, halogen, —CN, —NH₂, —OH, —NH(CH₃),—N(CH₃)₂, alkyl (including straight chain, branched and/or unsaturatedalkyl), substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, fluoro alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted alkoxy,fluoroalkoxy, —S-alkyl, S(═O)₂alkyl, —C(═O)NH[substituted orunsubstituted alkyl, or substituted or unsubstituted phenyl], —C(═O)N[Hor alkyl]₂, —OC(═O)N[substituted or unsubstituted alkyl]₂,—NHC(═O)NH[substituted or unsubstituted alkyl, or substituted orunsubstituted phenyl], —NHC(═O)alkyl, —N[substituted or unsubstitutedalkyl]C(═O)[substituted or unsubstituted alkyl], —NHC(═O)[substituted orunsubstituted alkyl], —C(OH)[substituted or unsubstituted alkyl]₂, and—C(NH₂)[substituted or unsubstituted alkyl]₂. In other embodiments, byway of example, an optional substituent is selected from oxo, fluorine,chlorine, bromine, iodine, —CN, —NH₂, —OH, —NH(CH₃), —N(CH₃)₂, —CH₃,—CH₂CH₃, —CH(CH₃)₂, —CF₃, —CH₂CF₃, —OCH₃, —OCH₂CH₃, —OCH(CH₃)₂, —OCF₃,—OCH₂CF₃, —S(═O)₂—CH₃, —C(═O)NH₂, —C(═O)—NHCH₃, —NHC(═O)NHCH₃,—C(═O)CH₃, and —C(═O)OH. In yet one embodiment, the substituents areindependently selected from the group consisting of C₁₋₆ alkyl, —OH,C₁₋₆ alkoxy, halo, amino, acetamido, oxo and nitro. In yet otherembodiments, the substituents are independently selected from the groupconsisting of C₁₋₆ alkyl, C₁₋₆ alkoxy, halo, acetamido, and nitro. Asused herein, where a substituent is an alkyl or alkoxy group, the carbonchain may be branched, straight or cyclic, with straight beingpreferred.

As used herein, the term “CT110” or “CT189” refers to1-(4-chlorophenyl)-3-(3-(4-fluorophenoxy)-prop-1-yl)guanidine).

As used herein, the term “haloperidol” or “Hal” refers to4-[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]-1-(4-fluorophenyl)butan-1-one.

As used herein, the term “IPAG” refers to1-(4-iodophenyl)-3-(2-adamantyl)guanidine).

As used herein, the term “PRE084” refers to 2-morpholin-4-ylethyl1-phenylcyclohexane-1-carboxylate.

As used herein, the term “S1RA” refers to4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl]morpholine.

As used herein, the term “SA4503” refers to1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl) piperazine.

Ranges: throughout this disclosure, various aspects of the disclosurecan be presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of thedisclosure. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub-ranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Compounds

The compounds contemplated within the disclosure may be synthesizedusing techniques well-known in the art of organic synthesis. Thestarting materials and intermediates required for the synthesis may beobtained from commercial sources or synthesized according to methodsknown to those skilled in the art.

In one aspect, the compound contemplated within the disclosure is acompound of formula (I), or a salt, solvate, or N-oxide thereof:

wherein:

ring A is a monocyclic or bicyclic aryl or a monocyclic or bicyclicheteroaryl ring, and wherein the aryl or heteroaryl ring is optionallysubstituted with 0-4 R¹ groups;

each occurrence of R¹ is independently selected from the groupconsisting of —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl, F,Cl, Br, I, —CN, —NO₂, —OR³, —SR³, —S(═O)R³, —S(═O)₂R³, —NHS(═O)₂R³,—C(═O)R³, —OC(═O)R³, —CO₂R³, —OCO₂R³, —CH(R³)₂, —N(R³)₂, —C(═O)N(R³)₂,—OC(═O)N(R³)₂, —NHC(═O)NH(R³), —NHC(═O)R³, —NHC(═O)OR³, —C(OH)(R³)₂, and—C(NH₂)(R³)₂;

each occurrence of R² is independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, and —C₁-C₃ alkyl-(C₃-C₆cycloalkyl), wherein the alkyl, heteroalkyl or cycloalkyl group isoptionally substituted with 0-5 R¹ groups, or X³ and R² combine to forma (C₃-C₇)heterocycloalkyl group, optionally substituted with 0-2 R¹groups;

each occurrence of R³ is independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, aryl, and —C₁-C₃alkyl-(C₃-C₆ cycloalkyl), wherein the alkyl, heteroalkyl, aryl, orcycloalkyl group is optionally substituted with 0-5 R¹ groups;

X¹ is —CH₂—, —S—, —O— or —(NR₂);

X² is ═CH₂, ═S, ═O or ═NR²; and

X³ is —S—, —O—, or —NR²—.

In certain embodiments, ring A is a monocyclic aryl or monocyclicheteroaryl ring optionally substituted with 0-4 R¹ groups. In otherembodiments, ring A is unsubstituted. In yet other embodiments, ring Ais phenyl or substituted phenyl.

In a preferred embodiment, X¹ and X³ are both —NH—, and X² is ═NH.

In another aspect, the compound contemplated within the disclosure is acompound of formula (II), or a salt, solvate, or N-oxide thereof:

R^(A)—R^(B)  (II),

wherein:

R^(A) is selected from the group consisting of

wherein

-   -   X⁴ is selected from the group consisting of methoxy, F, Cl, Br,        and I; and    -   R^(B) is selected from the group consisting of:

In another aspect, the compound contemplated within the disclosure is acompound of formula (III), or a salt, solvate, or N-oxide thereof:

wherein within formula (III);

each occurrence of R¹ and R² is independently selected from the groupconsisting of —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl, F,Cl, Br, I, —CN, —NO₂, —OR⁵, —SR⁵, —S(═O)R⁵, —S(═O)₂R⁵, —NHS(═O)₂R⁵,—C(═O)R⁵, —OC(═O)R⁵, —CO₂R⁵, —OCO₂R⁵, —CH(R⁵)₂, —N(R⁵)₂, —C(═O)N(R⁵)₂,—OC(═O)N(R⁵)₂, —NHC(═O)NH(R⁵), —NHC(═O)R⁵, —NHC(═O)OR⁵, —C(OH)(R⁵)₂, and—C(NH₂)(R⁵)₂;

R³ is selected from the group consisting of —C₁-C₆ alkyl, —C₁-C₆fluoroalkyl, —C₁-C₆ alkoxy, F, Cl, Br, and I;

R⁴ is selected from the group consisting of —C₁-C₆ alkyl, —C₁-C₆ alkoxy,F, Cl, Br, and I;

each occurrence of R⁵ is independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, aryl, and —C₁-C₃alkyl-(C₃-C₆ cycloalkyl), wherein the alkyl, heteroalkyl, aryl, orcycloalkyl group is optionally substituted.

X is selected from the group consisting of CH₂, C═O, or O;

n is an integer from 1-3;

x is an integer from 0-4; and

y is an integer from 0-4.

In certain embodiments, the compound contemplated within the disclosureis selected from the group consisting of:

-   1-(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A;    also known as JMS-51-58 or 51-58);-   1-(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (Compound    B);-   1-(n-propyl)-3-(4-iodophenyl)guanidine (Compound C);-   1-(n-propyl)-3-(4-methoxyphenyl)guanidine (Compound D);-   1,3-bis(3-(4-fluorophenoxy)propyl)guanidine (Compound E);-   1-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine    (Compound F);-   1-(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine (Compound    G);-   1-(3-(4-fluorophenoxy)propyl)-3-(4-methyl-2-oxo-2H-chromen-7-yl)guanidine    (Compound H);-   a salt, solvate or N-oxide thereof; and any combinations thereof.

Preparation of the Compounds

Compounds contemplated within the disclosure may be prepared by thegeneral schemes described herein, using the synthetic method known bythose skilled in the art. The following examples illustrate non-limitingembodiments of the disclosure.

In a non-limiting embodiment, the synthesis of unsymmetricalN,N′-disubstituted guanidines is accomplished by coupling an arylcyanamide and an amine. In certain embodiments, the coupling reactiontakes place at an elevated temperature ranging from 80° C. to 250° C. Ananiline may be converted to an aryl cyanamide with cyanogen bromide inether. The unsymmetrical N,N′-disubstituted guanidine is then formed bycoupling the aryl cyanamide with an amine. Non-limiting examples ofcoupling methods include heating in acetonitrile at reflux, and heatingat 120° C. in a microwave.

In another non-limiting embodiment, unsymmetrical N,N′-disubstitutedguanidines may be synthesized by coupling a benzimidothioate and anamine. For example, an aniline may be reacted with potassiumisothiocyanate to provide a thiourea. The thiourea may then be treatedwith methyl iodide in acetone heated to reflux, providing the desiredbenzimidothioate. The unsymmetrical N,N′-disubstituted guanidine maythen be formed by coupling the benzimidothioate with an amine. Anon-limiting example of a coupling method includes heating in ethanol atreflux.

The compounds of the disclosure may possess one or more stereocenters,and each stereocenter may exist independently in either the R or Sconfiguration. In certain embodiments, compounds described herein arepresent in optically active or racemic forms. It is to be understoodthat the compounds described herein encompass racemic, optically-active,regioisomeric and stereoisomeric forms, or combinations thereof thatpossess the therapeutically useful properties described herein.Preparation of optically active forms is achieved in any suitablemanner, including by way of non-limiting example, by resolution of theracemic form with recrystallization techniques, synthesis fromoptically-active starting materials, chiral synthesis, orchromatographic separation using a chiral stationary phase. In certainembodiments, a mixture of one or more isomer is utilized as thetherapeutic compound described herein. In other embodiments, compoundsdescribed herein contain one or more chiral centers. These compounds areprepared by any means, including stereoselective synthesis,enantioselective synthesis and/or separation of a mixture of enantiomersand/or diastereomers. Resolution of compounds and isomers thereof isachieved by any means including, by way of non-limiting example,chemical processes, enzymatic processes, fractional crystallization,distillation, and chromatography.

The methods and formulations described herein include the use ofN-oxides (if appropriate), crystalline forms (also known as polymorphs),solvates, amorphous phases, and/or pharmaceutically acceptable salts ofcompounds having the structure of any compound of the disclosure, aswell as metabolites and active metabolites of these compounds having thesame type of activity. Solvates include water, ether (e.g.,tetrahydrofuran, methyl tert-butyl ether) or alcohol (e.g., ethanol)solvates, acetates and the like. In certain embodiments, the compoundsdescribed herein exist in solvated forms with pharmaceuticallyacceptable solvents such as water, and ethanol. In other embodiments,the compounds described herein exist in unsolvated form.

In certain embodiments, the compounds of the disclosure may exist astautomers. All tautomers are included within the scope of the compoundspresented herein.

In certain embodiments, compounds described herein are prepared asprodrugs. A “prodrug” refers to an agent that is converted into theparent drug in vivo. In certain embodiments, upon in vivoadministration, a prodrug is chemically converted to the biologically,pharmaceutically or therapeutically active form of the compound. Inother embodiments, a prodrug is enzymatically metabolized by one or moresteps or processes to the biologically, pharmaceutically ortherapeutically active form of the compound.

In certain embodiments, sites on, for example, the aromatic ring portionof compounds of the disclosure are susceptible to various metabolicreactions. Incorporation of appropriate substituents on the aromaticring structures may reduce, minimize or eliminate this metabolicpathway. In certain embodiments, the appropriate substituent to decreaseor eliminate the susceptibility of the aromatic ring to metabolicreactions is, by way of example only, a deuterium, a halogen, or analkyl group.

Compounds described herein also include isotopically-labeled compoundswherein one or more atoms is replaced by an atom having the same atomicnumber, but an atomic mass or mass number different from the atomic massor mass number usually found in nature. Examples of isotopes suitablefor inclusion in the compounds described herein include and are notlimited to ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ³⁶Cl, ¹⁸F, ¹²³I, ¹²⁵I, ¹³N, ¹⁵N, ¹⁵O,¹⁷O, ¹⁸O, ³²P, and ³⁵S. In certain embodiments, isotopically-labeledcompounds are useful in drug and/or substrate tissue distributionstudies. In other embodiments, substitution with heavier isotopes suchas deuterium affords greater metabolic stability (for example, increasedin vivo half-life or reduced dosage requirements). In yet otherembodiments, substitution with positron emitting isotopes, such as ¹¹C,¹⁸F, ¹⁵O and ¹³N, is useful in Positron Emission Topography (PET)studies for examining substrate receptor occupancy. Isotopically-labeledcompounds are prepared by any suitable method or by processes using anappropriate isotopically-labeled reagent in place of the non-labeledreagent otherwise employed.

In certain embodiments, the compounds described herein are labeled byother means, including, but not limited to, the use of chromophores orfluorescent moieties, bioluminescent labels, or chemiluminescent labels.

The compounds described herein, and other related compounds havingdifferent substituents are synthesized using techniques and materialsdescribed herein and as described, for example, in Fieser & Fieser'sReagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons,1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991), Larock's Comprehensive OrganicTransformations (VCH Publishers Inc., 1989), March, Advanced OrganicChemistry 4^(th) Ed., (Wiley 1992); Carey & Sundberg, Advanced OrganicChemistry 4th Ed., Vols. A and B (Plenum 2000, 2001), and Green & Wuts,Protective Groups in Organic Synthesis 3rd Ed., (Wiley 1999) (all ofwhich are incorporated by reference for such disclosure). Generalmethods for the preparation of compound as described herein are modifiedby the use of appropriate reagents and conditions, for the introductionof the various moieties found in the formula as provided herein.

Compounds described herein are synthesized using any suitable proceduresstarting from compounds that are available from commercial sources, orare prepared using procedures described herein.

In certain embodiments, reactive functional groups, such as hydroxyl,amino, imino, thio or carboxy groups, are protected in order to avoidtheir unwanted participation in reactions. Protecting groups are used toblock some or all of the reactive moieties and prevent such groups fromparticipating in chemical reactions until the protective group isremoved. In other embodiments, each protective group is removable by adifferent means.

Protective groups that are cleaved under totally disparate reactionconditions fulfill the requirement of differential removal.

In certain embodiments, protective groups are removed by acid, base,reducing conditions (such as, for example, hydrogenolysis), and/oroxidative conditions. Groups such as trityl, dimethoxytrityl, acetal andt-butyldimethylsilyl are acid labile and are used to protect carboxy andhydroxy reactive moieties in the presence of amino groups protected withCbz groups, which are removable by hydrogenolysis, and Fmoc groups,which are base labile. Carboxylic acid and hydroxy reactive moieties areblocked with base labile groups such as, but not limited to, methyl,ethyl, and acetyl, in the presence of amines that are blocked with acidlabile groups, such as t-butyl carbamate, or with carbamates that areboth acid and base stable but hydrolytically removable.

In certain embodiments, carboxylic acid and hydroxy reactive moietiesare blocked with hydrolytically removable protective groups such as thebenzyl group, while amine groups capable of hydrogen bonding with acidsare blocked with base labile groups such as Fmoc. Carboxylic acidreactive moieties are protected by conversion to simple ester compoundsas exemplified herein, which include conversion to alkyl esters, or areblocked with oxidatively-removable protective groups such as2,4-dimethoxybenzyl, while co-existing amino groups are blocked withfluoride labile silyl carbamates.

Allyl blocking groups are useful in the presence of acid- andbase-protecting groups since the former are stable and are subsequentlyremoved by metal or pi-acid catalysts. For example, an allyl-blockedcarboxylic acid is deprotected with a palladium-catalyzed reaction inthe presence of acid labile t-butyl carbamate or base-labile acetateamine protecting groups. Yet another form of protecting group is a resinto which a compound or intermediate is attached. As long as the residueis attached to the resin, that functional group is blocked and does notreact. Once released from the resin, the functional group is availableto react.

Typically blocking/protecting groups may be selected from:

Other protecting groups, plus a detailed description of techniquesapplicable to the creation of protecting groups and their removal aredescribed in Greene & Wuts, Protective Groups in Organic Synthesis, 3rdEd., John Wiley & Sons, New York, N.Y., 1999, and Kocienski, ProtectiveGroups, Thieme Verlag, New York, N.Y., 1994, which are incorporatedherein by reference for such disclosure.

Methods

The disclosure includes a method of disrupting viral lifecycle,infection, and/or dissemination in a virus-infected subject, and/orpreventing or minimizing virus infection and/or dissemination in asubject, wherein the virus comprises at least one surface viral proteinthat is involved in viral entry and/or viral infection. The disclosurefurther includes a method of inhibiting, minimizing, and/or preventingformation of virus particles in a virus-infected subject, wherein thevirus comprises at least one surface viral protein that is involved inviral entry and/or viral infection. The disclosure further includes amethod of altering production, post-translational modification,assembly, maturation, and/or functional cell surface expression of atleast one virus protein involved in viral entry and/or viral infectionin a subject's cell. The disclosure further includes a method ofinitiating and/or stimulating selective autophagosomal, lysosomal,and/or proteasomal degradation of at least one virus protein involved inviral entry and/or viral infection in a subject's cell. The disclosurefurther includes a method of decreasing and/or inhibiting increase ofamount, concentration, and/or production of at least one virus proteininvolved in viral entry and/or viral infection in a subject's cell. Thedisclosure further includes a method of altering and/or disturbingsubcellular localization and/or virus promoting activity of at least onevirus protein involved in viral entry and/or viral infection in asubject's cell. The disclosure further includes a method of minimizingand/or preventing a surface viral protein incorporation into a virion ina virus-infected eukaryotic cell.

The disclosure further includes a method of promoting autophagicdegradation of NSP6 in a coronavirus-infected eukaryotic cell. Thedisclosure further includes a method of decreasing or inhibitingincrease of NSP6 concentration in a coronavirus-infected eukaryoticcell. The disclosure further includes a method of minimizing and/orsuppressing TMPRSS2 protein levels and cell surface localization on aneukaryotic cell.

In certain embodiments, the virus comprises a flavivirus. In otherembodiments, the flavivirus comprises at least one of Zika virus, Denguevirus, and Powassan virus. In yet other embodiments, the flaviviruscomprises at least one of Apoi virus, Aroa virus, Bamaga virus, Bagazavirus, Banzi virus, Bouboui virus, Bukalasa bat virus, Cacipacore virus,Carey Island virus, Cowbone Ridge virus, Dakar bat virus, Dengue virus,Edge Hill virus, Entebbe bat virus, Gadgets Gully virus, Ilheus virus,Israel turkey meningoencephalomyelitis virus, Japanese encephalitisvirus, Jugra virus, Jutiapa virus, Kadam virus, Kedougou virus, Kokoberavirus, Koutango virus, Kyasanur Forest disease virus, Langat virus,Louping ill virus, Meaban virus, Modoc virus, Montana myotisleukoencephalitis virus, Murray Valley encephalitis virus, Ntaya virus,Omsk hemorrhagic fever virus, Phnom Penh bat virus, Powassan virus, RioBravo virus, Royal Farm virus, Saboya virus, Saint Louis encephalitisvirus, Sal Vieja virus, San Perlita virus, Saumarez Reef virus, Sepikvirus, Tembusu virus, Tick-borne encephalitis virus, Tyuleniy virus,Uganda S virus, Usutu virus, Wesselsbron virus, West Nile virus, Yaoundevirus, Yellow fever virus, Yokose virus, and Zika virus

In certain embodiments, the virus comprises a Coronavirus. In otherembodiments, the Coronavirus comprises at least one of anAlphacoronavirus, a Betacoronavirus, a Gammacoronavirus, and aDeltacoronavirus. In yet other embodiments, the Coronavirus comprises atleast one of MERS-CoV, SARS-CoV, and SARS-CoV-2, and any variantsthereof.

In certain embodiments, the virus comprises a coronavirus and thesurface viral protein comprises a Spike (S) protein. In certainembodiments, the method comprises administering to the subject aneffective amount of at least one compound contemplated within thedisclosure. In other embodiments, the subject is further administered atleast one additional antiviral agent and/or at least one additionalagent that treats, ameliorates, and/or prevents one or more virusinfection symptoms or co-morbidities. In yet other embodiments, thecompound and the at least one additional agent are co-administered tothe subject. In yet other embodiments, the compound and the at least oneadditional agent are co-formulated.

In certain embodiments, the subject is a mammal. In other embodiments,the mammal is a human.

Combination Therapies

The compounds contemplated within the disclosure are intended to beuseful in combination with one or more additional compounds. Theseadditional compounds may comprise compounds of the present disclosureand/or at least one additional antiviral agent and/or at least oneadditional agent that treats one or more coronavirus infection symptomsor co-morbidities.

A synergistic effect may be calculated, for example, using suitablemethods such as, for example, the Sigmoid-E_(max) equation (Holford &Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the equation of Loeweadditivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol.114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv.Enzyme Regul. 22:27-55). Each equation referred to above may be appliedto experimental data to generate a corresponding graph to aid inassessing the effects of the drug combination. The corresponding graphsassociated with the equations referred to above are theconcentration-effect curve, isobologram curve and combination indexcurve, respectively.

Administration/Dosage/Formulations

The regimen of administration may affect what constitutes an effectiveamount. The therapeutic formulations contemplated within the disclosuremay be administered to the subject either prior to or after the onset ofa coronavirus infection. Further, several divided dosages, as well asstaggered dosages may be administered daily or sequentially, or the dosemay be continuously infused, or may be a bolus injection. Further, thedosages of the therapeutic formulations contemplated within thedisclosure may be proportionally increased or decreased as indicated bythe exigencies of the therapeutic or prophylactic situation.

Administration of the compositions contemplated within the disclosure toa patient, preferably a mammal, more preferably a human, may be carriedout using known procedures, at dosages and for periods of time effectiveto treat a coronavirus infection in the patient. An effective amount ofthe therapeutic compound necessary to achieve a therapeutic effect mayvary according to factors such as the state of the disease or disorderin the patient; the age, sex, and weight of the patient; and the abilityof the therapeutic compound contemplated within the disclosure to treata coronavirus infection in the patient. Dosage regimens may be adjustedto provide the optimum therapeutic response. For example, severaldivided doses may be administered daily or the dose may beproportionally reduced as indicated by the exigencies of the therapeuticsituation. A non-limiting example of an effective dose range for atherapeutic compound contemplated within the disclosure is from about 1and 5,000 mg/kg of body weight/per day. One of ordinary skill in the artwould be able to study the relevant factors and make the determinationregarding the effective amount of the therapeutic compound without undueexperimentation.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions contemplated within the disclosure may be varied so as toobtain an amount of the active ingredient that is effective to achievethe desired therapeutic response for a particular patient, composition,and mode of administration, without being toxic to the patient.

In particular, the selected dosage level depends upon a variety offactors including the activity of the particular compound employed, thetime of administration, the rate of excretion of the compound, theduration of the treatment, other drugs, compounds or materials used incombination with the compound, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well, known in the medical arts.

A medical doctor, e.g., physician or veterinarian, having ordinary skillin the art may readily determine and prescribe the effective amount ofthe pharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds contemplated within thedisclosure employed in the pharmaceutical composition at levels lowerthan that required in order to achieve the desired therapeutic effectand gradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulatethe compound in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the patients tobe treated; each unit containing a predetermined quantity of therapeuticcompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical vehicle. The dosage unitforms contemplated within the disclosure are dictated by and directlydependent on (a) the unique characteristics of the therapeutic compoundand the particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding/formulating such atherapeutic compound for the treatment of a coronavirus infection in apatient.

In certain embodiments, the compositions of the disclosure areformulated using one or more pharmaceutically acceptable excipients orcarriers. In certain embodiments, the pharmaceutical compositions of thedisclosure comprise a therapeutically effective amount of a compound ofthe disclosure and a pharmaceutically acceptable carrier.

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms may be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it is preferable to include isotonic agents, for example, sugars,sodium chloride, or polyalcohols such as mannitol and sorbitol, in thecomposition. Prolonged absorption of the injectable compositions may bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate or gelatin. In certainembodiments, the pharmaceutically acceptable carrier is not DMSO alone.

In certain embodiments, the compositions of the disclosure areadministered to the patient in dosages that range from one to five timesper day or more. In other embodiments, the compositions of thedisclosure are administered to the patient in range of dosages thatinclude, but are not limited to, once every day, every two, days, everythree days to once a week, and once every two weeks. It is readilyapparent to one skilled in the art that the frequency of administrationof the various combination compositions of the disclosure varies fromindividual to individual depending on many factors including, but notlimited to, age, disease or disorder to be treated, gender, overallhealth, and other factors. Thus, the disclosure should not be construedto be limited to any particular dosage regime and the precise dosage andcomposition to be administered to any patient is determined by theattending physical taking all other factors about the patient intoaccount.

Compounds of the disclosure for administration may be in the range offrom about 1 g to about 10,000 mg, about 20 μg to about 9,500 mg, about40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg toabout 7,500 mg, about 200 μg to about 7,000 mg, about 3050 μg to about6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg toabout 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80mg to about 500 mg, and any and all whole or partial incrementstherebetween.

In some embodiments, the dose of a compound of the disclosure is fromabout 1 mg and about 2,500 mg. In some embodiments, a dose of a compoundof the disclosure used in compositions described herein is less thanabout 10,000 mg, or less than about 8,000 mg, or less than about 6,000mg, or less than about 5,000 mg, or less than about 3,000 mg, or lessthan about 2,000 mg, or less than about 1,000 mg, or less than about 500mg, or less than about 200 mg, or less than about 50 mg. Similarly, insome embodiments, a dose of a second compound as described herein isless than about 1,000 mg, or less than about 800 mg, or less than about600 mg, or less than about 500 mg, or less than about 400 mg, or lessthan about 300 mg, or less than about 200 mg, or less than about 100 mg,or less than about 50 mg, or less than about 40 mg, or less than about30 mg, or less than about 25 mg, or less than about 20 mg, or less thanabout 15 mg, or less than about 10 mg, or less than about 5 mg, or lessthan about 2 mg, or less than about 1 mg, or less than about 0.5 mg, andany and all whole or partial increments thereof.

In certain embodiments, the present disclosure is directed to a packagedpharmaceutical composition comprising a container holding atherapeutically effective amount of a compound of the disclosure, aloneor in combination with a second pharmaceutical agent; and instructionsfor using the compound to treat, prevent, or reduce one or more symptomsof Sigma-receptor related disorders or diseases in a patient.

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for oral, parenteral, nasal, intravenous,subcutaneous, enteral, or any other suitable mode of administration,known to the art. The pharmaceutical preparations may be sterilized andif desired mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure buffers, coloring, flavoring and/or aromatic substances and thelike. They may also be combined where desired with other active agents,e.g., other analgesic agents.

Routes of administration of any of the compositions of the disclosureinclude oral, nasal, rectal, intravaginal, parenteral, buccal,sublingual or topical. The compounds for use in the disclosure may beformulated for administration by any suitable route, such as for oral orparenteral, for example, transdermal, transmucosal (e.g., sublingual,lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- andperivaginally), (intra)nasal and (trans)rectal), intravesical,intrapulmonary, intraduodenal, intragastrical, intrathecal,subcutaneous, intramuscular, intradermal, intra-arterial, intravenous,intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets,capsules, caplets, pills, gel caps, troches, dispersions, suspensions,solutions, syrups, granules, beads, transdermal patches, gels, powders,pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs,suppositories, liquid sprays for nasal or oral administration, drypowder or aerosolized formulations for inhalation, compositions andformulations for intravesical administration and the like. It should beunderstood that the formulations and compositions that would be usefulin the present disclosure are not limited to the particular formulationsand compositions that are described herein.

Oral Administration

For oral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules, caplets and gelcaps. Thecompositions intended for oral use may be prepared according to anymethod known in the art and such compositions may contain one or moreagents selected from the group consisting of inert, non-toxicpharmaceutically excipients that are suitable for the manufacture oftablets. Such excipients include, for example an inert diluent such aslactose; granulating and disintegrating agents such as cornstarch;binding agents such as starch; and lubricating agents such as magnesiumstearate. The tablets may be uncoated or they may be coated by knowntechniques for elegance or to delay the release of the activeingredients. Formulations for oral use may also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertdiluent.

For oral administration, the compounds of the disclosure may be in theform of tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,polyvinylpyrrolidone, hydroxypropylcellulose orhydroxypropylmethylcellulose); fillers (e.g., cornstarch, lactose,microcrystalline cellulose or calcium phosphate); lubricants (e.g.,magnesium stearate, talc, or silica); disintegrates (e.g., sodium starchglycollate); or wetting agents (e.g., sodium lauryl sulphate). Ifdesired, the tablets may be coated using suitable methods and coatingmaterials such as OPADRY™ film coating systems available from Colorcon,West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-PType, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White,32K18400). Liquid preparation for oral administration may be in the formof solutions, syrups or suspensions. The liquid preparations may beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifying agent (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily estersor ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid).

The present disclosure also includes a multi-layer tablet comprising alayer providing for the delayed release of one or more compounds of thedisclosure, and a further layer providing for the immediate release ofanother medication. Using a wax/pH-sensitive polymer mix, a gastricinsoluble composition may be obtained in which the active ingredient isentrapped, ensuring its delayed release.

Parenteral Administration

For parenteral administration, the compounds of the disclosure may beformulated for injection or infusion, for example, intravenous,intramuscular or subcutaneous injection or infusion, or foradministration in a bolus dose and/or continuous infusion. Suspensions,solutions or emulsions in an oily or aqueous vehicle, optionallycontaining other formulatory agents such as suspending, stabilizingand/or dispersing agents may be used.

Additional Administration Forms

Additional dosage forms of this disclosure include dosage forms asdescribed in U.S. Pat. Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389;5,582,837; and 5,007,790. Additional dosage forms of this disclosurealso include dosage forms as described in U.S. Patent Applications Nos.20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and20020051820. Additional dosage forms of this disclosure also includedosage forms as described in PCT Applications Nos. WO 03/35041; WO03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404; WO02/32416; WO 01/97783; WO 01/56544; WO 01/32217; WO 98/55107; WO98/11879; WO 97/47285; WO 93/18755; and WO 90/11757.

Controlled Release Formulations and Drug Delivery Systems

In certain embodiments, the formulations of the present disclosure maybe, but are not limited to, short-term, rapid-offset, as well ascontrolled, for example, sustained release, delayed release andpulsatile release formulations.

The term sustained release is used in its conventional sense to refer toa drug formulation that provides for gradual release of a drug over anextended period of time, and that may, although not necessarily, resultin substantially constant blood levels of a drug over an extended timeperiod. The period of time may be as long as a month or more and shouldbe a release which is longer that the same amount of agent administeredin bolus form.

For sustained release, the compounds may be formulated with a suitablepolymer or hydrophobic material which provides sustained releaseproperties to the compounds. As such, the compounds for use the methodof the disclosure may be administered in the form of microparticles, forexample, by injection or in the form of wafers or discs by implantation.

In certain embodiments of the disclosure, the compounds of thedisclosure are administered to a patient, alone or in combination withanother pharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense torefer to a drug formulation that provides for an initial release of thedrug after some delay following drug administration and that mat,although not necessarily, includes a delay of from about 10 minutes upto about 12 hours.

The term pulsatile release is used herein in its conventional sense torefer to a drug formulation that provides release of the drug in such away as to produce pulsed plasma profiles of the drug after drugadministration.

The term immediate release is used in its conventional sense to refer toa drug formulation that provides for release of the drug immediatelyafter drug administration.

As used herein, short-term refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes and any or all whole orpartial increments thereof after drug administration after drugadministration.

As used herein, rapid-offset refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes, and any and all whole orpartial increments thereof after drug administration.

Dosing

The therapeutically effective amount or dose of a compound of thepresent disclosure depends on the age, sex and weight of the patient,the current medical condition of the patient and the progression ofSigma-receptor related disorders or diseases in the patient beingtreated. The skilled artisan is able to determine appropriate dosagesdepending on these and other factors.

A suitable dose of a compound of the present disclosure may be in therange of from about 0.01 mg to about 5,000 mg per day, such as fromabout 0.1 mg to about 1,000 mg, for example, from about 1 mg to about500 mg, such as about 5 mg to about 250 mg per day. The dose may beadministered in a single dosage or in multiple dosages, for example from1 to 4 or more times per day. When multiple dosages are used, the amountof each dosage may be the same or different. For example, a dose of 1 mgper day may be administered as two 0.5 mg doses, with about a 12-hourinterval between doses.

It is understood that the amount of compound dosed per day may beadministered, in non-limiting examples, every day, every other day,every 2 days, every 3 days, every 4 days, or every 5 days. For example,with every other day administration, a 5 mg per day dose may beinitiated on Monday with a first subsequent 5 mg per day doseadministered on Wednesday, a second subsequent 5 mg per day doseadministered on Friday, and so on.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the modulator of the disclosure isoptionally given continuously; alternatively, the dose of drug beingadministered is temporarily reduced or temporarily suspended for acertain length of time (i.e., a “drug holiday”). The length of the drugholiday optionally varies between 2 days and 1 year, including by way ofexample only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days,12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days,120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days,320 days, 350 days, or 365 days. The dose reduction during a drugholiday includes from 10%-100%, including, by way of example only, 10%,15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, is reduced, as a function of theviral load, to a level at which the improved disease is retained. Incertain embodiments, patients require intermittent treatment on along-term basis upon any recurrence of symptoms and/or infection.

The compounds for use in the method of the disclosure may be formulatedin unit dosage form. The term “unit dosage form” refers to physicallydiscrete units suitable as unitary dosage for patients undergoingtreatment, with each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect,optionally in association with a suitable pharmaceutical carrier. Theunit dosage form may be for a single daily dose or one of multiple dailydoses (e.g., about 1 to 4 or more times per day). When multiple dailydoses are used, the unit dosage form may be the same or different foreach dose.

Toxicity and therapeutic efficacy of such therapeutic regimens areoptionally determined in cell cultures or experimental animals,including, but not limited to, the determination of the LD₅₀ (the doselethal to 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between the toxicand therapeutic effects is the therapeutic index, which is expressed asthe ratio between LD₅₀ and ED₅₀. Capsid assembly modulators exhibitinghigh therapeutic indices are preferred. The data obtained from cellculture assays and animal studies are optionally used in formulating arange of dosage for use in human. The dosage of such capsid assemblymodulators lies preferably within a range of circulating concentrationsthat include the ED₅₀ with minimal toxicity. The dosage optionallyvaries within this range depending upon the dosage form employed and theroute of administration utilized.

Those skilled in the art recognizes, or is able to ascertain using nomore than routine experimentation, numerous equivalents to the specificprocedures, embodiments, claims, and examples described herein. Suchequivalents were considered to be within the scope of this disclosureand covered by the claims appended hereto. For example, it should beunderstood, that modifications in reaction conditions, including but notlimited to reaction times, reaction size/volume, and experimentalreagents, such as solvents, catalysts, pressures, atmosphericconditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents,with art-recognized alternatives and using no more than routineexperimentation, are within the scope of the present application.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present disclosure.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

The examples described herein illustrate aspects of the presentdisclosure. However, they are in no way a limitation of the teachings ordisclosure of the present disclosure as set forth herein.

The examples described herein are provided for purposes of illustrationonly, and are not intended to be limiting unless otherwise specified.Thus, the disclosure should in no way be construed as being limited tothe examples described herein, but rather, should be construed toencompass any and all variations which become evident as a result of theteaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the description and illustrative examples, makeand utilize the compounds of the present disclosure and practice theclaimed methods. The working examples therefore, specifically point outselected embodiments of the present disclosure, and are not to beconstrued as limiting in any way the remainder of the disclosure.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this disclosure has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis disclosure may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the disclosure. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

EXAMPLES

The disclosure is now described with reference to the followingExamples. These Examples are provided for the purpose of illustrationonly, and the disclosure is not limited to these Examples, but ratherencompasses all variations that are evident as a result of the teachingsprovided herein.

Example 1

Coronaviruses (CoV) replicate in the cytoplasm of host cells and in theprocess co-opt cellular membranes and machinery to produce and assemblenew infectious viral particles for dissemination throughout the infectedorganism. Initiation of CoV replication is completely dependent on theER. Conversely, the CoV life cycle and associated activities have aprofound impact on ER function. Viral replication rapidly producessubstantial amounts of viral proteins that are co- orpost-translationally inserted into the ER and targeted to the site ofparticle assembly, largely in the ER-Golgi intermediate compartment(ERGIC). Assembled viral particles in the ERGIC are transported to thecell surface via the secretory pathway and infectious virions releasedfrom the host cell by exocytosis.

SARS-CoV-2 Spike (S) Protein.

The spike (S) protein that forms protrusions on the surface of maturevirions gives coronaviruses their characteristic crown-like appearancewhen viewed by electron microscopy. The S protein mediates selectivevirus entry into host cells, is the primary determinant of tissuetropism and viral host range, and is a major trigger of host immuneresponses. Infection and cytopathic effects (CPE) do not occur in theabsence of functional S protein. Many viruses, CoVs included, readilyadapt to new environments through mutation and recombination of their Sprotein (or equivalent). This ability to adapt can broaden the hostrange to promote cross-species infection and is intrinsic to manyviruses. Therefore, neutralizing antibodies tend to lose their efficacybecause of changes in the S protein receptor binding domain. In the caseof CoVs, sera from recovered SARS 2003 and COVID19 patients whose Sproteins share significant homology show limited cross-neutralization.Additionally, a recently discovered mutation in the S protein was linkedto increased virulence and proposed as a contributing factor todifferences in virulence and mortality between SARS-CoV-2 viral cladeson the West and East Coast of the U.S.

Receptor binding and membrane fusion are the initial and critical stepsin CoV infection. The CoV S glycoprotein is a typical class I viralfusion protein that consists of two principal components: an ectodomainconsisting of a receptor-binding subunit S1, a single-pass transmembranedomain containing a membrane-fusion subunit S2. During virus entry, S1of SARS-CoV and CoV-2 binds to the host cell surface receptorangiotensin converting enzyme 2 (ACE2). ACE2 binding destabilizes theprefusion Spike resulting in shedding of the S1 subunit and S2 mediatedfusion of the host and viral membranes, subsequently allowing viralgenomes to enter host cells. Protease cleavage is required foractivation of the fusion potential of S glycoprotein of SARS-CoV-2 Sprotein and related viruses, SARS-CoV and MERS-CoV. In the case ofSARS-CoV and CoV-2, Spike is cleaved by transmembrane protease serineprotease-2 (TMPRSS2) in lung epithelial cells.

Transmembrane Protease Serine Protease-2 (TMPRSS2).

SARS-CoV and -CoV-2 Spike are activated by TMPRSS2; enhanced TMPRSS2expression promotes increased infectivity in both in vitro and in vivomouse models of SARS-CoV lung infection. TMPRSS2 is an androgen-induced,androgen receptor (AR) target gene which is a putative contributingfactor to the apparently increased virulence of COVID19 in men, althoughit remains unclear if this holds true in a broader population analysis.In addition to its role in the secretory pathway, Sigma1 regulates ARsignaling and that Sigma1 modulators can block TMPRSS2 induction byandrogens (FIGS. 1A-1D).

Example 2: Sigma1/SIGMAR1, TMPRSS2, and ACE2 Expression in Lung AlveolarType II (AT2) Epithelial Cells

SIGMAR1/Sigma1 is abundantly expressed in the lung (FIGS. 2A-2B).Although a role for SIGMAR1/Sigma1 in lung physiology andpathophysiology has not been clearly established, Sigma1 may play a rolein lung inflammation and stress leading to fibrosis. A consequence ofthe secretory pathway regulating properties of Sigma1 modulators is thatthey can also be used to elicit immune modulatory and anti-inflammatoryeffects. Particularly high expression of ACE2 and TMPRSS2 transcriptswere observed specifically in lung alveolar type II (AT2) cells with asignificant correlation between transcripts of the two genes (FIGS. 2B &2D). Analysis of the dataset showed that SIGMAR1 is also highly enrichedin AT2 cells and has a highly significant correlation with TMPRSS2 andACE2 expression (FIGS. 2B-2C).

Example 3: Identification of Small Molecule Sigma1 Modulators thatSuppress Infectivity of Replication Competent SARS-CoV-2

In certain embodiments, compounds that bind Sigma1 and trigger ER stresscan block SARS-CoV-2 replication, as measured by quantitative qRT-PCRassay of viral infection and replication and associated cytopathiceffects (CPE) assessed by an in vitro plaque assay.

A discrete set of Sigma1 modulators was tested in an assay ofreplication competent SARS-CoV-2 infectivity. Only Sigma1 inhibitorsthat induce ER stress (i.e., IPAG, CT189, and haloperidol) suppressedSARS-CoV-2 infection, in rank order of ER stress inducing potency.Strikingly, IPAG and CT189 blocked SARS-CoV-2 infection by 1,000 (IPAG,CT189) fold. Haloperidol had a clear but modest antiviral effect. Incontrast, Sigma1 activators (SA4503 and PRE084), which not induce ERstress, did not block infection. S1RA, a putative antagonist that doesnot induce ER stress, did not block infection (FIG. 3 ).

A pharmacophore and hypothesis driven screen of Sigma1 modulatorcompounds is performed in an in vitro infectivity assay usingreplication competent SARS-CoV-2 virus. Changes in the status of SIGMAR1transcript and Sigma1 protein in infected cells are monitored to probethe role of Sigma1 in the infected host cell and in viral replication.

Infection Assays.

Infectivity assays are performed using replication competent SARS-CoV-2virus. Compounds with diverse Sigma1 pharmacology (including thosetested in Huh7.5 cell lines in FIG. 3 ) that bind Sigma1 but inducedistinct molecular actions are screened. Traditional plaque/cytopathiceffect (CPE) assays are performed using replication competent infectiousSARS-COV-2 virus to identify compounds that block infection with ananti-viral TCID₅₀ of <10 μM. Standard CoV induced plaque assays andqRTPCR based viral titer calculations are performed. VeroE6 (ATCCCRL-1586), and VeroE6/TMPRSS2 (stably transfected to express high levelsof TMPRSS2) are used to model differences in response by susceptible andhighly susceptible cells to CoV-2 infection, replication, and CPE.VeroE6 cells are a widely used, standard model cell line for CoV studiesand express high levels of angiotensin converting enzyme 2 (ACE2), thecognate receptor for SARS-CoV and SARS-CoV-2. In certain embodiments,these compounds are antiviral in primary respiratory epithelial cells.

SIGMAR1/Sigma1 Status in CoV Infected Cells.

In certain embodiments, if Sigma1 is acutely engaged and utilized duringviral replication, biomarkers of Sigma1 involvement in viral replicationmight include upregulation of SIGMAR1 mRNA transcripts and Sigma1protein as well as altered subcellular distribution of Sigma1 to reflectengagement by viral proteins. To evaluate these potential biomarkers,SIGMAR1 mRNA transcript levels are monitored by qRT-PCR, and Sigma1protein levels and distribution patterns are monitored by IB andimmunocytochemical (ICC) staining in replication competent CoV-2infected cells in vitro. Differences in levels and/or localization ofSigma1 may reflect differential protein-interactions, which can beevaluated with protein-pulldown and proximity ligation assays todetermine likely changes in Sigma1 associated protein complexes relevantto viral replication.

Example 4: Characterization of Impact of Sigma1 Modulators on SARS-CoV-2Spike (S) Protein and Host Cell TMPRSS2

In certain embodiments, Sigma1 targeting agents that trigger prolongedER stress can block the maturation of and eliminate the SARS-CoV-2 Sprotein and/or decrease TMPRSS2 levels to suppress viral infection. Keymechanisms by which efficacious antiviral Sigma1 modulators blockSARS-CoV-2 viral entry can be identified and characterized. SARS-CoV-2Spike is activated by TMPRSS2 whose enhanced expression promotesinfectivity in vitro and in vivo. As Spike and TMPRSS2 are integralmembrane glycoproteins with multiple N-glycosylation sites, they areboth dependent on the ER for translation and post-translationalmodification (PTM) for maturation and transport to the cell surface.

The functional impact of Sigma1 modulators on S protein inducedcell-to-cell fusion is evaluated when expressed independently in Vero E6cells. Changes in S protein incorporation into pseudovirions, and thusthe impact of Sigma1 modulators on production of infectious viralparticle, are also evaluated. For S protein studies, the followingplasmid constructs can be used: untagged parental SARS-CoV-2 S protein;3×FLAG tagged S protein described by Ou et al., 2020, Naturecommunications 11:1620; HA-tagged S protein described by Hoffman et al.,2020, Cell, doi:10.1016/j.cell.2020.02.052. TMRPSS2 focused assays canbe used to determine levels and localization of protein, cell-to-cellfusion efficiency and infectivity of Sigma1 modulator treated VeroE6 andVeroE6/TMPRSS2 host cells.

Western Blot Data Support Antiviral Mechanism of Action of SpecificSigma1-Targeting Compounds.

Only certain Sigma1 inhibitors, those that induce ER stress andautophagy (IPAG, CT189), inhibit SARS-CoV-2 infection, while a putativeSigma1 antagonist that does not induce ER stress or autophagy (S1RA)does not block infection (FIG. 1 ). As shown in FIG. 4 , treatment ofhuman Huh7.5 cells with these Sigma1 selective inhibitors resulted in astriking decrease in SARS-CoV-2 Spike protein 48 hours post-infection,while treatment with SIRA (which binds Sigma1 with high affinity butdoes not trigger ER stress and autophagy) did not. Importantly toxicityin these cells was not observed at the concentrations tested.

Impact of Sigma1 Modulation on Spike and TMPRSS2 Mediated Cell-to-CellFusion.

This assay is a direct measure of S protein-receptor binding andfusogenic activity. Cell surface expression of S protein, independent ofother viral proteins, can trigger the fusion of cells expressing Sprotein at their surface with cells expressing its cognate receptor,ACE2. Subsequent to binding its receptor ACE2, S protein's fusogenicactivity is triggered by co-expressed TMPRSS2-mediated cleavage on thesurface of the host cell. In this assay, expression of S proteintriggered cell-to-cell fusion can induce the formation of multinucleatedcellular syncytia, the readout of common cell fusion assays. EvaluatingTMPRSS2 is important here because the SARS-CoV and -CoV-2 S proteins areproteolytically activated by TMPRSS2 and saliently increases SARS-CoVand SARS-CoV-2 replication and syncytium formation in vitro and in vivo.

This assay can be performed using VeroE6 cells and VeroE6/TMPRSS2 cellswhich over express TMPRSS2 and that are highly susceptible to SARS-CoV-2infection and promote virus-host cell membrane fusion independent ofendocytosis. This assay essentially can be performed as describedelsewhere (Hoffmann, et al., 2020, Cell, doi:10.1016/j.cell.2020.02.052)with minor modifications. A beta-galactosidase reporter plasmid wastransfected into the VeroE6 cells previously published to detect singlefusion events as well as larger syncytia (Kim, et al., 2003, J. Virol.77:963-969). Sigma1 shRNA knockdown studies are also conducted in thesecells to confirm that Sigma1 is required for expression, maturation,localization, and fusogenic activity of S protein. Cell-to-cell fusionof VeroE6 and VeroE6/TMPRSS2 cells treated with Sigma1 modulators and/orin which Sigma1 is knocked down with shRNA are compared.

Sigma1 Inhibitor Blocks the Formation of Syncytia in Cells ExpressingSARS-CoV-2 Spike and TMPRSS2.

SARS-CoV-2 infected cells fuse with neighboring cells to form syncytia,mediated by the viral spike protein interacting with the cellular ACE2receptor and enhanced by the host cellular protease, TMPRSS2. VeroE6cells are transfected with plasmids expressing SARS-CoV-2 spike andhuman TMPRSS2 and the number of syncytia formed (indicator of frequencyof cell-cell fusion events) and the number of nuclei per syncytia(indicator of magnitude of Spike triggered cell fusion and potentialcytopathic effect) are measured in DMSO and compound treated cells.Compounds of interest show a decrease in both the number and the size ofsyncytia compound-treated cells, compared to controls. In certainnon-limiting embodiments, certain Sigma1 inhibitors can inhibitinfection by targeting the CoV-2 Spike protein and host cell TMPRSS2 fordegradation. Protein lysates from the cells in the syncytia assays areimmunoblotted for Spike and TMPRSS2. A decrease in both Spike andTMPRSS2 protein is observed in compound-treated cells.

Spike and TMPRSS2 Protein Levels, Localization, and Post-TranslationalModification.

In certain embodiments, Sigma1 modulators can selectively triggerproteasomal and autolysosomal degradation of membrane associated andintegral membrane proteins. Changes in total cellular S protein levelscan be evaluated by immunoblot. Changes in S protein glycosylation areevaluated by performing EndoH and PNGase F sensitivity assays. Sigma1modulator induced changes to S protein cell surface expression can beevaluated by surface biotinylation/pulldown assay and orthogonally byconfocal microscopy. Using VeroE6 and VeroE6/TMRPSS2 cells, it can betested whether Sigma1 modulators decrease TMPRSS2 protein levels byproteasomal or autolysosomal degradation and if their maturation isblocked by disruption of N-glycosylation in ER.

Incorporation of SARS-CoV-2 S Protein into Pseudotyped Viral Particles.

This assay can test impact of Sigma1 modulators on S glycoproteinmaturation and incorporation into pseudovirions. Pseudovirions can beproduced using a lentiviral vector and packaging system with SARS-CoV-2S glycoprotein. Pseudovirions can be produced by co-transfecting 293Tcells with psPAX2, pLenti-CMVGFP/Luc, and SARS-CoV-2 S plasmids. Theefficiency of SARS-CoV-2 S protein incorporation into lentiviralpseudovirions canl be evaluated by immunoblot of isolated viralparticles from the cell culture medium.

Example 5: Determining Whether Sigma1 Modulators Restrict SARS-CoV-2Infection in Primary Cells and a Syrian Golden Hamster Model ofInfection

Sigma1 modulators can inhibit SARS-CoV-2 infection in vitro using cellline models. A repertoire of primary lung cells are tested, andantiviral efficacy of Sigma1 modulators are evaluated, in vivo, in aSyrian hamster model of SARS-CoV-2 infection and pathology.

SIGMAR1 expression is elevated in AT2 cells and this correlates with theexpression of ACE2 and TMPRSS2 (FIGS. 2A-2D). It is assessed whetherSigma1 modulators are antiviral in commercially-available primary airwaycells, beginning with human AT2 cells (Accegen Biotech) and humaniPSC-derived alveolar epithelial cells (Cell Applications). Thesestudies are extended to primary nasal epithelial (Epithelix), bronchialepithelial (Lonza), human small airway epithelial (Lonza), andbronchial/tracheal epithelial cells (ATCC). Without wishing to belimited by any theory, it is important to test cells from differentairways regions as CoV-2 can infect diverse tissue in people. Thesecells are cultured in air-liquid interface (ALI) cultures, which mimicthe airway.

Syrian golden hamsters are a useful small animal model for both SARS-CoVand SARS-CoV-2 infection. CoV-2 replicates efficiently in the lungs ofthese animals and causes pathological lesions similar to those ofCOVID19 patients with pneumonia. Additionally, CoV-2 was transmittedfrom infected to naive hamsters via aerosols, providing further supportthat this model is similar to human infection. Sigma1 modulators aretested in this model of infection to further evaluate the possibility ofusing these molecules as therapeutic agents in SARS-CoV-2 infection andpathology.

Infections of Syrian Golden Hamsters with SARS-CoV-2.

Syrian golden hamsters can be with Sigma1-targeting agents to determinewhether these compounds are antiviral in vivo. The Syrian golden hamstermodel for SARS-CoV-2 infection was successfully established. FIG. 5illustrates robust detection of SARS-CoV-2 nucleocapsid protein in thelungs of Syrian golden hamsters at 3 days post-infection, which is notpresent in uninfected controls. These data demonstrate that this modelcan be used to test the activity of Sigma1-targeting compounds in vivo.

Antiviral Activity of Sigma1 Modulators in Primary Airway Cells.

Cells are treated with compounds of interest at a concentration of 10 μMfor 2 hours, followed by infection with SARS-CoV-2 at an MOI of 1. After24, 48 and 72 hours, the media is aspirated, cells are collected inTrizol, RNA is extracted, and infection is measured by qRT-PCR usingprimers specific to the SARS-CoV-2 genome. For any compound that hasantiviral activity, dose-response assays are perform to determine theIC₅₀ and CC₅₀ in these cultures. In addition to assessing infection byqPCR, viruses released from the apical side of the ALI cultures are alsocollected and viral titers assessed by TCID₅₀ assays. An importantcritical factor in evaluating the value of Sigma1 modulators astherapeutic agents for SARS-CoV-2 infection is to determine theireffectiveness relative to the timing of viral infection, as these can beadministered subsequent to infection. Therefore, it is evaluated whetherthe addition of Sigma1 modulators after viral infection in primary humancells is effective. Primary cells are infected with SARS-CoV-2 at an MOIof 1 and cells treated with Sigma1 modulators at 6, 12, and 24 h hourpost-infection; viral infection is assessed by qRT-PCR. For allexperiments, the expression of SIGMAR1, TMPRSS2, and ACE2 mRNA ismonitored using qRT-PCR. Also monitored are the protein expression andlocalization of Sigma1, TMPRSS2 and ACE2 via immunoblotting andimmunocytochemistry (ICC), respectively.

Ability of Sigma1 Modulators to Restrict SARS-CoV-2 Replication in aHamster Model of Infection.

To determine if Sigma1 modulators are antiviral in a Syrian goldenhamster model of infection, hamsters are treated with compounds thatrestrict CoV-2 infection in primary cells. The animals are administered10 and 30 mg/kg of each compound or drug vehicle by i.p. injection theday before viral infection. Ten hamsters are used per group, with anequal number of males and females. All animals are implanted withmicrochips to allow to monitor body temperature over the course of theexperiment (UID Identification Solutions). Hamsters are infected withCoV-2 (TCID₅₀ of 105), delivered intranasally. At days 2, 4, and 8 afterthe infection, animals are euthanized, and nasal turbinates, lungs,livers, spleen, and kidneys are isolated. Nasal turbinates and one lungare used for extraction of the challenge virus for subsequent virustitration and virus quantification by qRT-PCR, as well as forquantitation of SIGMAR1 and TMPRSS2. Remaining lung is used forhistological characterization of disease and for IHC staining of CoV-2antigens. For the other organs, viral loads are determined by qRT-PCR toanalyze the spread of the virus. To test whether Sigma1 modulators areantiviral when administered after infection, these experiments areperformed as described elsewhere herein, with the exception that thecompounds are administered at days 2 and 4 after infection, witheuthanasia at 6 and 8 days. In certain embodiments, by combiningbiochemical MOA assays with in vitro and in vivo antiviral efficacyassays (infectivity and CPE), one can identify antiviral Sigma1targeting compounds that have therapeutic utility.

Example 6

FIG. 6 illustrates the finding that compounds of the disclosure inhibitDengue virus infection. U2OS cells were treated with DMSO and 10 μM ofCT110 (also known as CT189). 2 hours after starting treatment, U2OScells were infected with a multiplicity of infection (MOI) of 1 forDengue virus and incubated at 37° C. for ˜16-18 hours. Cells wereharvested with Trizol and RNA was extracted for qPCR. Y-axis indicatesfold change in infection.

FIG. 7 illustrates the finding that compounds of the disclosure inhibitZika virus infection. U2OS cells were treated with DMSO and 10 μM ofCT110 (also known as CT189). 2 hours after starting treatment, U2OScells were infected with a multiplicity of infection (MOI) of 1 for Zikavirus and incubated at 37° C. for ˜16-18 hours. Cells were harvestedwith Trizol and RNA was extracted for qPCR. Y-axis indicates fold changein infection.

FIG. 8 illustrates the finding that compounds of the disclosure inhibitPowassan virus infection. U2OS cells were treated with DMSO and 10 μM ofCT110 (also known as CT189). 2 hours after starting treatment, U20Scells were infected with a multiplicity of infection (MOI) of 1 forPowassan virus and incubated at 37° C. for ˜16-18 hours. Cells wereharvested with Trizol and RNA was extracted for qPCR. Y-axis indicatesfold change in infection.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this disclosure has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this disclosure may be devised by others skilled in theart without departing from the true spirit and scope of the disclosure.The appended claims are intended to be construed to include all suchembodiments and equivalent variations.

1. A method of disrupting virus lifecycle, infection, or disseminationin a virus-infected subject, or preventing and/or minimizing virusinfection or dissemination in a subject, wherein the virus comprises atleast one surface viral protein that is involved in viral entry or viralinfection, the method comprising administering to the subject atherapeutically effective amount of at least one compound selected fromthe group consisting of: (i) a compound of Formula (I):

wherein: ring A is a monocyclic or bicyclic aryl or a monocyclic orbicyclic heteroaryl ring, and wherein the aryl or heteroaryl ring isoptionally substituted with 0-4 R¹ groups; each occurrence of R¹ isindependently selected from the group consisting of —C₁-C₆ alkyl, —C₁-C₆fluoroalkyl, —C₁-C₆ heteroalkyl, F, Cl, Br, I, —CN, —NO₂, —OR³, —SR³,—S(═O)R³, —S(═O)₂R³, —NHS(═O)₂R³, —C(═O)R³, —OC(═O)R³, —CO₂R³, —OCO₂R³,—CH(R³)₂, —N(R³)₂, —C(═O)N(R³)₂, —OC(═O)N(R³)₂, —NHC(═O)NH(R³),—NHC(═O)R³, —NHC(═O)OR³, —C(OH)(R³)₂, and —C(NH₂)(R³)₂; each occurrenceof R² is independently selected from the group consisting of H, C₁-C₆alkyl, C₁-C₆ heteroalkyl, and —C₁-C₃ alkyl-(C₃-C₆ cycloalkyl), whereinthe alkyl, heteroalkyl or cycloalkyl group is optionally substitutedwith 0-5 R¹ groups, or X³ and R² combine to form a(C₃-C₇)heterocycloalkyl group, optionally substituted with 0-2 R¹groups; each occurrence of R³ is independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, aryl, and —C₁-C₃alkyl-(C₃-C₆ cycloalkyl), wherein the alkyl, heteroalkyl, aryl, orcycloalkyl group is optionally substituted with 0-5 R¹ groups; X¹ is—CH₂—, —S—, —O— or —(NR²)—; X² is ═CH₂, ═S, ═O or ═NR²; and X³ is —S—,—O—, or —NR²—; and (ii) a compound of Formula (II):R^(A)—R^(B)  (II), wherein: R^(A) is selected from the group consistingof

X⁴ is selected from the group consisting of methoxy, F, Cl, Br, and I;and R^(B) is selected from the group consisting of:

(iii) a compound of formula (III):

wherein: each occurrence of R¹ and R² is independently selected from thegroup consisting of —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆heteroalkyl, F, Cl, Br, I, —CN, —NO₂, —OR⁵, —SR⁵, —S(═O)R⁵, —S(═O)₂R⁵,—NHS(═O)₂R⁵, —C(═O)R⁵, —OC(═O)R⁵, —CO₂R⁵, —OCO₂R⁵, —CH(R⁵)₂, —N(R⁵)₂,—C(═O)N(R⁵)₂, —OC(═O)N(R⁵)₂, —NHC(═O)NH(R⁵), —NHC(═O)R⁵, —NHC(═O)OR⁵,—C(OH)(R⁵)₂, and —C(NH₂)(R⁵)₂; R³ is selected from the group consistingof —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ alkoxy, F, Cl, Br, and I; R⁴is selected from the group consisting of —C₁-C₆ alkyl, —C₁-C₆ alkoxy, F,Cl, Br, and I; each occurrence of R⁵ is independently selected from thegroup consisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, aryl, and —C₁-C₃alkyl-(C₃-C₆ cycloalkyl), wherein the alkyl, heteroalkyl, aryl, orcycloalkyl group is optionally substituted; X is selected from the groupconsisting of CH₂, C═O, or O; n is an integer from 1-3; x is an integerfrom 0-4; and y is an integer from 0-4; a salt, solvate, or N-oxidethereof; and any combinations thereof.
 2. A method of inhibiting,minimizing, or preventing formation of virus particles in avirus-infected subject, wherein the virus comprises at least one surfaceviral protein that is involved in viral entry or viral infection, themethod comprising administering to the subject a therapeuticallyeffective amount of at least one compound selected from the groupconsisting of: (i) a compound of Formula (I):

wherein: ring A is a monocyclic or bicyclic aryl or a monocyclic orbicyclic heteroaryl ring, and wherein the aryl or heteroaryl ring isoptionally substituted with 0-4 R¹ groups; each occurrence of R¹ isindependently selected from the group consisting of —C₁-C₆ alkyl, —C₁-C₆fluoroalkyl, —C₁-C₆ heteroalkyl, F, Cl, Br, I, —CN, —NO₂, —OR³, —SR³,—S(═O)R³, —S(═O)₂R³, —NHS(═O)₂R³, —C(═O)R³, —OC(═O)R³, —CO₂R³, —OCO₂R³,—CH(R³)₂, —N(R³)₂, —C(═O)N(R³)₂, —OC(═O)N(R³)₂, —NHC(═O)NH(R³),—NHC(═O)R³, —NHC(═O)OR³, —C(OH)(R³)₂, and —C(NH₂)(R³)₂; each occurrenceof R² is independently selected from the group consisting of H, C₁-C₆alkyl, C₁-C₆ heteroalkyl, and —C₁-C₃ alkyl-(C₃-C₆ cycloalkyl), whereinthe alkyl, heteroalkyl or cycloalkyl group is optionally substitutedwith 0-5 R¹ groups, or X³ and R² combine to form a(C₃-C₇)heterocycloalkyl group, optionally substituted with 0-2 R¹groups; each occurrence of R³ is independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, aryl, and —C₁-C₃alkyl-(C₃-C₆ cycloalkyl), wherein the alkyl, heteroalkyl, aryl, orcycloalkyl group is optionally substituted with 0-5 R¹ groups; X¹ is—CH₂—, —S—, —O— or —(NR²)—; X² is ═CH₂, ═S, ═O or ═NR²; and X³ is —S—,—O—, or —NR²—; and (ii) a compound of Formula (II):R^(A)—R  (II), wherein: R^(A) is selected from the group consisting of

X⁴ is selected from the group consisting of methoxy, F, Cl, Br, and I;and R^(B) is selected from the group consisting of:

(iii) a compound of formula (III):

wherein: each occurrence of R¹ and R² is independently selected from thegroup consisting of —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆heteroalkyl, F, Cl, Br, I, —CN, —NO₂, —OR⁵, —SR⁵, —S(═O)R⁵, —S(═O)₂R⁵,—NHS(═O)₂R⁵, —C(═O)R⁵, —OC(═O)R⁵, —CO₂R⁵, —OCO₂R⁵, —CH(R⁵)₂, —N(R⁵)₂,—C(═O)N(R⁵)₂, —OC(═O)N(R⁵)₂, —NHC(═O)NH(R⁵), —NHC(═O)R⁵, —NHC(═O)OR⁵,—C(OH)(R⁵)₂, and —C(NH₂)(R⁵)₂; R³ is selected from the group consistingof —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ alkoxy, F, Cl, Br, and I; R⁴is selected from the group consisting of —C₁-C₆ alkyl, —C₁-C₆ alkoxy, F,Cl, Br, and I; each occurrence of R⁵ is independently selected from thegroup consisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, aryl, and —C₁-C₃alkyl-(C₃-C₆ cycloalkyl), wherein the alkyl, heteroalkyl, aryl, orcycloalkyl group is optionally substituted; X is selected from the groupconsisting of CH₂, C═O, or O; n is an integer from 1-3; x is an integerfrom 0-4; and y is an integer from 0-4; a salt, solvate, or N-oxidethereof; and any combinations thereof.
 3. A method of (i) alteringproduction, post-translational modification, assembly, maturation, orfunctional cell surface expression of at least one virus proteininvolved in viral entry or viral infection in a subject's cell; (ii)initiating or stimulating selective autophagosomal, lysosomal, orproteasomal degradation of at least one virus protein involved in viralentry or viral infection in a subject's cell; (iii) decreasing orinhibiting increase of amount, concentration, or production of at leastone virus protein involved in viral entry or viral infection in asubject's cell; (iv) altering or disturbing subcellular localization orvirus promoting activity of at least one virus protein involved in viralentry or viral infection in a subject's cell; or (v) minimizing orpreventing incorporation of a surface viral protein into a virion in avirus-infected eukaryotic cell; the method comprising administering tothe subject a therapeutically effective amount of at least one compoundselected from the group consisting of: (i) a compound of Formula (I):

wherein: ring A is a monocyclic or bicyclic aryl or a monocyclic orbicyclic heteroaryl ring, and wherein the aryl or heteroaryl ring isoptionally substituted with 0-4 R¹ groups; each occurrence of R¹ isindependently selected from the group consisting of —C₁-C₆ alkyl, —C₁-C₆fluoroalkyl, —C₁-C₆ heteroalkyl, F, Cl, Br, I, —CN, —NO₂, —OR³, —SR³,—S(═O)R³, —S(═O)₂R³, —NHS(═O)₂R³, —C(═O)R³, —OC(═O)R³, —CO₂R³, —OCO₂R³,—CH(R³)₂, —N(R³)₂, —C(═O)N(R³)₂, —OC(═O)N(R³)₂, —NHC(═O)NH(R³),—NHC(═O)R³, —NHC(═O)OR³, —C(OH)(R³)₂, and —C(NH₂)(R³)₂; each occurrenceof R² is independently selected from the group consisting of H, C₁-C₆alkyl, C₁-C₆ heteroalkyl, and —C₁-C₃ alkyl-(C₃-C₆ cycloalkyl), whereinthe alkyl, heteroalkyl or cycloalkyl group is optionally substitutedwith 0-5 R¹ groups, or X³ and R² combine to form a(C₃-C₇)heterocycloalkyl group, optionally substituted with 0-2 R¹groups; each occurrence of R³ is independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, aryl, and —C₁-C₃alkyl-(C₃-C₆ cycloalkyl), wherein the alkyl, heteroalkyl, aryl, orcycloalkyl group is optionally substituted with 0-5 R¹ groups; X¹ is—CH₂—, —S—, —O— or —(NR²)—; X² is ═CH₂, ═S, ═O or ═NR²; and X³ is —S—,—O—, or —NR²—; and (ii) a compound of Formula (II):R^(A)—R  (II), wherein: R^(A) is selected from the group consisting of

X⁴ is selected from the group consisting of methoxy, F, Cl, Br, and I;and R^(B) is selected from the group consisting of:

(iii) a compound of formula (III):

wherein: each occurrence of R¹ and R² is independently selected from thegroup consisting of —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆heteroalkyl, F, Cl, Br, I, —CN, —NO₂, —OR⁵, —SR⁵, —S(═O)R⁵, —S(═O)₂R⁵,—NHS(═O)₂R⁵, —C(═O)R⁵, —OC(═O)R⁵, —CO₂R⁵, —OCO₂R⁵, —CH(R⁵)₂, —N(R⁵)₂,—C(═O)N(R⁵)₂, —OC(═O)N(R⁵)₂, —NHC(═O)NH(R⁵), —NHC(═O)R⁵, —NHC(═O)R⁵,—C(OH)(R⁵)₂, and —C(NH₁₂)(R⁵)₂; R³ is selected from the group consistingof —C₁-C₆ fluoroalkyl, —C₁-C₆ alkoxy, F, Cl, Br, and I; R⁴ is selectedfrom the group consisting of —C₁-C₆ alkyl, —C₁-C₆ alkoxy, F, Cl, Br, andI; each occurrence of R⁵ is independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, aryl, and —C₁-C₃alkyl-(C₃-C₆ cycloalkyl), wherein the alkyl, heteroalkyl, aryl, orcycloalkyl group is optionally substituted; X is selected from the groupconsisting of CH₂, C═O, or O; n is an integer from 1-3; x is an integerfrom 0-4; and y is an integer from 0-4; a salt, solvate, or N-oxidethereof; and any combinations thereof.
 4. The method of claim 1, whereinthe virus comprises a flavivirus or a Coronavirus.
 5. The method ofclaim 4, wherein the virus comprises the flavivirus, and wherein theflavivirus comprises at least one of Apoi virus, Aroa virus, Bamagavirus, Bagaza virus, Banzi virus, Bouboui virus, Bukalasa bat virus,Cacipacore virus, Carey Island virus, Cowbone Ridge virus, Dakar batvirus, Dengue virus, Edge Hill virus, Entebbe bat virus, Gadgets Gullyvirus, Ilheus virus, Israel turkey meningoencephalomyelitis virus,Japanese encephalitis virus, Jugra virus, Jutiapa virus, Kadam virus,Kedougou virus, Kokobera virus, Koutango virus, Kyasanur Forest diseasevirus, Langat virus, Louping ill virus, Meaban virus, Modoc virus,Montana myotis leukoencephalitis virus, Murray Valley encephalitisvirus, Ntaya virus, Omsk hemorrhagic fever virus, Phnom Penh bat virus,Powassan virus, Rio Bravo virus, Royal Farm virus, Saboya virus, SaintLouis encephalitis virus, Sal Vieja virus, San Perlita virus, SaumarezReef virus, Sepik virus, Tembusu virus, Tick-borne encephalitis virus,Tyuleniy virus, Uganda S virus, Usutu virus, Wesselsbron virus, WestNile virus, Yaounde virus, Yellow fever virus, Yokose virus, and Zikavirus.
 6. (canceled)
 7. The method of claim 4, wherein the viruscomprises the Coronavirus, and at least one of the following applies:(a) the Coronavirus comprises at least one of an Alphacoronavirus, aBetacoronavirus, a Gammacoronavirus, and a Deltacoronavirus, (b) theCoronavirus comprises at least one of MERS-CoV, SARS-CoV, andSARS-CoV-2, (c) the at least one surface viral protein comprises NSP6 ora S glycoprotein.
 8. (canceled)
 9. (canceled)
 10. The method of claim 1,wherein at least one of the following applies: (the compound is selectedfrom the group consisting of:1-(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A);1-(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (Compound B);1-(n-propyl)-3-(4-iodophenyl)guanidine (Compound C);1-(n-propyl)-3-(4-methoxyphenyl)guanidine (Compound D);1-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine(Compound F); 1-(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine(Compound G); a salt, solvate or N-oxide thereof, and any combinationsthereof, (b) the compound is selected from the group consisting of:1,3-bis(3-(4-fluorophenoxy)propyl)guanidine (Compound E);1-(3-(4-fluorophenoxy)propyl)-3-(4-methyl-2-oxo-2H-chromen-7-yl)guanidine)(Compound H); a salt, solvate or N-oxide thereof, and any combinationsthereof, (c) the compound is administered as a pharmaceuticalcomposition further comprising a pharmaceutically acceptable carrier,(d) the subject is further administered at least one additionalantiviral agent or at least one agent that treats one or more virusinfection symptoms or co-morbidities, (e) the compound is administeredby a route comprising oral, nasal, rectal, intravaginal, parenteral,buccal, sublingual or topical, (f) the subject is a mammal, (g) thesubject is a human. 11-16. (canceled)
 17. The method of claim 2, whereinthe virus comprises a flavivirus or a Coronavirus.
 18. The method ofclaim 17, wherein the virus comprises the flavivirus, and wherein theflavivirus comprises at least one of Apoi virus, Aroa virus, Bamagavirus, Bagaza virus, Banzi virus, Bouboui virus, Bukalasa bat virus,Cacipacore virus, Carey Island virus, Cowbone Ridge virus, Dakar batvirus, Dengue virus, Edge Hill virus, Entebbe bat virus, Gadgets Gullyvirus, Ilheus virus, Israel turkey meningoencephalomyelitis virus,Japanese encephalitis virus, Jugra virus, Jutiapa virus, Kadam virus,Kedougou virus, Kokobera virus, Koutango virus, Kyasanur Forest diseasevirus, Langat virus, Louping ill virus, Meaban virus, Modoc virus,Montana myotis leukoencephalitis virus, Murray Valley encephalitisvirus, Ntaya virus, Omsk hemorrhagic fever virus, Phnom Penh bat virus,Powassan virus, Rio Bravo virus, Royal Farm virus, Saboya virus, SaintLouis encephalitis virus, Sal Vieja virus, San Perlita virus, SaumarezReef virus, Sepik virus, Tembusu virus, Tick-borne encephalitis virus,Tyuleniy virus, Uganda S virus, Usutu virus, Wesselsbron virus, WestNile virus, Yaounde virus, Yellow fever virus, Yokose virus, and Zikavirus.
 19. The method of claim 17, wherein the virus comprises theCoronavirus, and at least one of the following applies: (a) theCoronavirus comprises at least one of an Alphacoronavirus, aBetacoronavirus, a Gammacoronavirus, and a Deltacoronavirus, (b) theCoronavirus comprises at least one of MERS-CoV, SARS-CoV, andSARS-CoV-2, (c) the at least one surface viral protein comprises NSP6 ora S glycoprotein.
 20. The method of claim 2, wherein at least one of thefollowing applies: (a) the compound is selected from the groupconsisting of: 1-(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine(Compound A); 1-(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine(Compound B); 1-(n-propyl)-3-(4-iodophenyl)guanidine (Compound C);1-(n-propyl)-3-(4-methoxyphenyl)guanidine (Compound D);1-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine(Compound F); 1-(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine(Compound G); a salt, solvate or N-oxide thereof, and any combinationsthereof, (b) the compound is selected from the group consisting of:1,3-bis(3-(4-fluorophenoxy)propyl)guanidine (Compound E);1-(3-(4-fluorophenoxy)propyl)-3-(4-methyl-2-oxo-2H-chromen-7-yl)guanidine)(Compound H); a salt, solvate or N-oxide thereof, and any combinationsthereof, (c) the compound is administered as a pharmaceuticalcomposition further comprising a pharmaceutically acceptable carrier,(d) the subject is further administered at least one additionalantiviral agent or at least one agent that treats one or more virusinfection symptoms or co-morbidities, (e) the compound is administeredby a route comprising oral, nasal, rectal, intravaginal, parenteral,buccal, sublingual or topical, (f) the subject is a mammal, (g) thesubject is a human.
 21. The method of claim 3, wherein the viruscomprises a flavivirus or a Coronavirus.
 22. The method of claim 21,wherein the virus comprises the flavivirus, and wherein the flaviviruscomprises at least one of Apoi virus, Aroa virus, Bamaga virus, Bagazavirus, Banzi virus, Bouboui virus, Bukalasa bat virus, Cacipacore virus,Carey Island virus, Cowbone Ridge virus, Dakar bat virus, Dengue virus,Edge Hill virus, Entebbe bat virus, Gadgets Gully virus, Ilheus virus,Israel turkey meningoencephalomyelitis virus, Japanese encephalitisvirus, Jugra virus, Jutiapa virus, Kadam virus, Kedougou virus, Kokoberavirus, Koutango virus, Kyasanur Forest disease virus, Langat virus,Louping ill virus, Meaban virus, Modoc virus, Montana myotisleukoencephalitis virus, Murray Valley encephalitis virus, Ntaya virus,Omsk hemorrhagic fever virus, Phnom Penh bat virus, Powassan virus, RioBravo virus, Royal Farm virus, Saboya virus, Saint Louis encephalitisvirus, Sal Vieja virus, San Perlita virus, Saumarez Reef virus, Sepikvirus, Tembusu virus, Tick-borne encephalitis virus, Tyuleniy virus,Uganda S virus, Usutu virus, Wesselsbron virus, West Nile virus, Yaoundevirus, Yellow fever virus, Yokose virus, and Zika virus.
 23. The methodof claim 21, wherein the virus comprises the Coronavirus, and at leastone of the following applies: (a) the Coronavirus comprises at least oneof an Alphacoronavirus, a Betacoronavirus, a Gammacoronavirus, and aDeltacoronavirus, (b) the Coronavirus comprises at least one ofMERS-CoV, SARS-CoV, and SARS-CoV-2, (c) the at least one virus proteincomprises NSP6 or a S glycoprotein.
 24. The method of claim 3, whereinat least one of the following applies: (a) the compound is selected fromthe group consisting of:1-(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A);1-(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (Compound B);1-(n-propyl)-3-(4-iodophenyl)guanidine (Compound C);1-(n-propyl)-3-(4-methoxyphenyl)guanidine (Compound D);1-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine(Compound F); 1-(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine(Compound G); a salt, solvate or N-oxide thereof, and any combinationsthereof, (b) the compound is selected from the group consisting of:1,3-bis(3-(4-fluorophenoxy)propyl)guanidine (Compound E);1-(3-(4-fluorophenoxy)propyl)-3-(4-methyl-2-oxo-2H-chromen-7-yl)guanidine)(Compound H); a salt, solvate or N-oxide thereof, and any combinationsthereof, (c) the compound is administered as a pharmaceuticalcomposition further comprising a pharmaceutically acceptable carrier,(d) the subject is further administered at least one additionalantiviral agent or at least one agent that treats one or more virusinfection symptoms or co-morbidities, (e) the compound is administeredby a route comprising oral, nasal, rectal, intravaginal, parenteral,buccal, sublingual or topical, (f) the subject is a mammal, (g) thesubject is a human.